Mixtures of HMOs

ABSTRACT

The invention relates to a mixture of human milk oligosaccharides that consists essentially of:
         a) a component A which is 3-FL or DFL,
           a component B which is LNT, LNnT, LNFP-I or 2′-FL,   a component C, which is
               LNFP-II when component B is LNT, or   LNFP-III when component B is LNnT, or   LNDFH-I when component B is LNFP-I, or   DFL when component B is 2′-FL, and   
               a component D, which is
               lactose when component A is 3-FL, or   2′-FL when component A is DFL,   
               
           with the proviso that if component B is 2′-FL, then component A is 3-FL;
 
or consists essentially of:
   b) 3-FL,
           a component E which is LNT, LNnT or LNFP-I, and   a component F, which is
               LNFP-II when component E is LNT, or   LNFP-III when component E is LNnT, or   LNDFH-I when component E is LNFP-I,
 
and to processes for producing them and their uses.

FIELD OF THE INVENTION

The present invention relates to mixtures of Human Milk Oligosaccharides(“HMOs”), a process for making the mixtures, and applications of themixtures in human health.

BACKGROUND OF THE INVENTION

HMOs have become the subject of much interest in recent years due totheir roles in numerous biological processes occurring in the humanorganism. Mammalian milk contains at least 130 of these complexoligosaccharides (Urashima et al, Milk Oligosaccharides, Nova BiomedicalBooks, New York, 2011, ISBN: 978-1-61122-831-1).

Previously, the only source of HMOs had been mammalian milk whichcontains mostly water, together with 55-70 g/l lactose, 24-59 g/llipids, ca. 13 g/l proteins, 5-15 g/l HMOs and ca. 1.5 g/l minerals.

However, efforts to develop processes for synthesizing theseoligosaccharides have increased significantly in the last ten years dueto their roles in numerous human biological processes. In this regard,processes have been developed for producing HMOs by microbialfermentations, enzymatic processes, chemical syntheses, or combinationsof these technologies. For example, by chemical processes, LNnT can bemade as described in WO 2011/100980 and WO 2013/044928, LNT can besynthesized as described in WO 2012/155916 and WO 2013/044928, a mixtureof LNT and LNnT can be made as described in WO 2013/091660, 2′-FL can bemade as described in WO 2010/115934 and WO 2010/115935, 3-FL can be madeas described in WO 2013/139344, and 6′-SL and salts thereof can be madeas described in WO 2010/100979. As examples of biotechnologicalprocesses, WO 01/04341 and WO 2007/101862 describe how to make corehuman milk oligosaccharides optionally substituted by fucose or sialicacid using genetically modified E. coli. WO 2012/158517 and WO2013/154725 describe prebiotic compositions containing 3-FL, 2′-FL andDFL and WO 2012/112777 describes a mixture of 3-FL, 2′-FL, DFL andlactose produced in a genetically modified E. coli. As an example ofenzymatic processes, sialylated oligosaccharides can be made asdescribed in EP-A-577580.

Efforts have also been made to develop processes for synthesizingenzymatically mixtures of HMO oligosaccharides, without having tosynthesize all of the component oligosaccharides of the mixture asdescribed in WO 2012/156897 and WO 2012/156898. Such processes haveprovided reaction mixtures containing a plurality of differentoligosaccharides.

However, better processes have been sought for the synthesis of mixturesof HMOs.

Evidence is accumulating that the resident community of microbes, calledthe microbiome, in the human digestive tract plays a major role inhealth and disease. When the normal composition of the microbiome isthrown off balance, the human host can suffer consequences. Recentresearch has implicated microbiome imbalances in disorders as diverse ascancer, obesity, inflammatory bowel disease, psoriasis, asthma, andpossibly even autism. HMOs are believed to positively modulate themicrobiome, and they are of increasing interest for this purpose.However, the remarkable diversity of HMOs, coupled with their lack ofavailability, has hampered studies of the specific functions ofindividual HMOs. There is a clear need for specific HMOs or combinationsof HMOs to modulate the microbiome in a desired manner, so as to addressspecific human health issues.

SUMMARY OF THE INVENTION

A first aspect of this invention relates to a mixture of HMOs consistingessentially of

-   -   a component A which is 3-FL or DFL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL,    -   a component C, which is:        -   LNFP-II when component B is LNT, or        -   LNFP-III when component B is LNnT, or        -   LNDFH-I when component B is LNFP-I, or        -   DFL when component B is 2′-FL, and    -   a component D, which is:        -   lactose when component A is 3-FL, or        -   2′-FL when component A is DFL,            with the proviso that if component B is 2′-FL then component            A is 3-FL. Preferably in the HMO mixture of the first            aspect, component A is DFL.

A second aspect of this invention relates to a mixture of HMOsconsisting essentially of

-   -   3-FL,    -   a component E which is LNT, LNnT or LNFP-I, and    -   a component F, which is:        -   LNFP-II when component E is LNT, or        -   LNFP-III when component E is LNnT, or        -   LNDFH-I when component E is LNFP-I.

A third aspect of this invention relates to a process for making amixture of HMOs according to the first aspect, by reacting a component Aand a component B in the presence of an α1-3/4 transfucosidase toproduce a reaction mixture containing the components A, B, C and D, andthen removing the α1-3/4 transfucosidase from the reaction mixture.

A fourth aspect of this invention relates to a process for obtaining amixture of HMOs consisting essentially of:

-   -   3-FL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL, and    -   a component C, which is        -   LNFP-II when component C is LNT, or        -   LNFP-III when component C is LNnT, or        -   LNDFH-I when component C is LNFP-I, or        -   DFL when component C is 2′-FL,            preferably a mixture of the second aspect, comprising the            steps of reacting 3-FL and a component B in the presence of            an α1-3/4 transfucosidase to produce a reaction mixture            containing 3-FL, lactose and the components B and C, and            then removing lactose and the α1-3/4 transfucosidase from            the reaction mixture.

A fifth aspect of this invention relates to an anti-infectivecomposition for treating bacterial infections. The compositioncomprises, preferably consists essentially of:

-   -   DFL,    -   a component E which is LNT, LNnT or LNFP-I,    -   a component F, which is:        -   LNFP-II when component E is LNT, or        -   LNFP-III when component E is LNnT, or        -   LNDFH-I when component E is LNFP-I, and    -   2′-FL,        or    -   3-FL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL, and    -   a component C, which is:        -   LNFP-II when component B is LNT, or        -   LNFP-III when component B is LNnT, or        -   LNDFH-I when component B is LNFP-I, or        -   DFL when component B is 2′-FL;            or the composition consists essentially of:    -   3-FL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL,    -   a component C, which is:        -   LNFP-II when component B is LNT, or        -   LNFP-III when component B is LNnT, or        -   LNDFH-I when component B is LNFP-I, or        -   DFL when component B is 2′-FL, and    -   lactose.

Each of these anti-infective compositions contains a plurality ofdifferent HMOs with novel combinations of properties and biologicalactivities. Specifically, each composition can increase Bifidobacteriumabundance and Barnesiella abundance in the microbiome of a human.Preferably, each composition can reduce Firmicutes abundance in thehuman microbiome, especially Clostridia. Each composition can also beused to treat and/or reduce the risk of a broad range of bacterialinfections of a human.

A sixth aspect of this invention relates to a method of modulating themicrobiome of a human to increase Bifidobacterium abundance andBarnesiella abundance, preferably by reducing Firmicutes abundance,especially Clostridia.

A seventh aspect of this invention relates to a method of preventing ortreating bacterial infections in a human, especially antibioticresistant bacterial infections.

The methods of the sixth and seventh aspects each compriseadministering, to the human:

-   i) a composition comprising, preferably consisting essentially of:    -   3-FL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL, and    -   a component C, which is        -   LNFP-II when component B is LNT, or        -   LNFP-III when component B is LNnT, or        -   LNDFH-I when component B is LNFP-I, or        -   DFL when component B is 2′-FL;            or-   ii) a composition consisting essentially of:    -   3-FL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL, and    -   a component C, which is        -   LNFP-II when component B is LNT, or        -   LNFP-III when component B is LNnT, or        -   LNDFH-I when component B is LNFP-I, or        -   DFL when component B is 2′-FL; and    -   lactose;-   or iii) a composition comprising, preferably consisting essentially    of,    -   DFL,    -   a component E which is LNT, LNnT or LNFP-I,    -   a fucosylated component F, which is        -   LNFP-II when component E is LNT, or        -   LNFP-III when component E is LNnT, or        -   LNDFH-I when component E is LNFP-I, and    -   2′-FL.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with this invention, it has been surprisingly discoveredthat mixture of HMOs can provide an anti-infective composition fortreating bacterial infections through specific modulation of themicrobiome. The mixture increases Bifidobacterium abundance andBarnesiella abundance of the microbiome. The mixture also reducesFirmicutes abundance of the microbiome; especially Clostridia. Humanshaving increased abundance of Bifidobacterium and Barnesiella in theirmicrobiome are more resistant to a broad range of infections and recovermore quickly from these infections, including from antibiotic resistantinfections. It is believed that this improved resistance is attributableto the changes in the microbiome. The reduction in Firmicutes in themicrobiome especially Clostridia, attributable to this mixture, impartsadditional health benefits.

The First Aspect of the Invention

The first aspect is a mixture of HMOs consisting essentially of

-   -   a component A which is 3-FL or DFL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL,    -   a component C, which is        -   LNFP-II when component B is LNT, or        -   LNFP-III when component B is LNnT, or        -   LNDFH-I when component B is LNFP-I, or        -   DFL when component B is 2′-FL, and    -   a component D, which is        -   lactose when component A is 3-FL, or        -   2′-FL when component A is DFL,            with the proviso that if component B is 2′-FL then component            A is 3-FL.

In a preferred embodiment of the mixture of this first aspect, componentA is 3-FL. More preferably the molar ratio of (3-FL+component B)relative to the component C is 0.8-9.50, and the molar ratio of lactoseto the component C is about 1. Even more preferably, the molar ratio of3-FL to the component B is 0.07-7.7. Particularly in a mixtureconsisting essentially of 3-FL, 2′-FL, DFL and lactose, the molar ratioof 3-FL to 2′-FL is 0.05-7.7.

In a further preferred embodiment of the mixture of this first aspect,component A is 3-FL and component B is LNT or LNnT. Preferably, one ofthe molar ratios of 3-FL to the component C and the component B to thecomponent C is not more than 2 .

According to this first aspect, especially preferred are:

-   -   i) a mixture of HMOs which consists essentially of 3-FL, LNT,        LNFP-II and lactose and in which:        -   the molar ratio of (3-FL+LNT) relative to LNFP-II is            0.8-9.5, and        -   the molar ratio of lactose relative to LNFP-II is about 1,        -   provided that one of the molar ratios 3-FL to LNFP-II and            LNT to LNFP-II is not more than 2; and    -   ii) a mixture of HMOs which consists essentially of 3-FL, LNnT,        LNFP-III and lactose, and in which:        -   the molar ratio of (3-FL+LNnT) relative to LNFP-III is            0.8-9.5, and        -   the molar ratio of lactose relative to LNFP-III is about 1,        -   provided that one of the molar ratios 3-FL to LNFP-III and            LNnT to LNFP-III is not more than 2.

In a further preferred embodiment of the mixture of the first aspect,component A is 3-FL and component B is LNFP-I or 2′-FL. Preferably, oneof the molar ratios of 3-FL to the component C and of the component B tothe component C is not more than 2, and/or the molar ratio of 3-FL tothe component B is 0.07-7.7.

Especially preferred is a mixture of HMOs which consists essentially of3-FL, LNFP-I, LNDFH-I and lactose. Preferably, in this HMO mixture:

-   -   the molar ratio of (3-FL+LNFP-I) relative to LNDFH-I is 0.8-9.5,        and    -   the molar ratio of lactose relative to LNDFH-I is about 1;        and more preferably in this mixture:    -   one of the molar ratios of 3-FL to LNDFH-I and LNFP-I to LNDFH-I        is not more than 2.

Also according to this first aspect, especially preferred is a mixtureof HMOs which consists essentially of 3-FL, 2′-FL, DFL and lactose.Preferably, in this mixture:

-   -   the molar ratio of (3-FL+2′-FL) relative to DFL is 0.8-9.5 and    -   the molar ratio of lactose relative to DFL is about 1.

More preferably, this mixture has a molar ratio of 3-FL to 2′-FL of0.05-7.7 or 0.07-7.7. Also more preferably, the molar ratio of 3-FL toDFL or of 2′-FL to DFL is not more than 2.

In still another preferred embodiment of the mixture of the firstaspect, component A is DFL. Preferably, the molar ratio of the componentC relative to (DFL+component B) is at least 1:10, preferably at least1:7, more preferably at least 1:5, even more preferably at least 1:3.Also preferably, the molar ratio of DFL relative to the component B is0.17-6, preferably 0.25-4, more preferably about 1.

The mixture of this first aspect can consist essentially of 2′-FL, DFL,LNT and LNFP-II. This HMO mixture preferably has a molar ratio ofLNFP-II relative to (DFL+LNT) of at least 1:10, more preferably at least1:7, even more preferably at least 1:5, especially 1:3. This HMO mixturealso preferably has a molar ratio of DFL relative to LNT of 0.17-6,preferably 0.25-4, more preferably about 1. Also preferably, thismixture has a molar ratio of 2′-FL relative to LNFP-II of about 1.

The mixture of the first aspect can also consist essentially of 2′-FL,DFL, LNnT and LNFP-III. This HMO mixture preferably has a molar ratio ofLNFP-III relative to (DFL+LNnT) of at least 1:10, more preferably atleast 1:7, even more preferably at least 1:5, especially 1:3. This HMOmixture also preferably has a molar ratio of DFL relative to LNnT of0.17-6, preferably 0.25-4, more preferably about 1. Also preferably,this mixture has a molar ratio of 2′-FL relative to LNFP-III of about 1.

The mixture of the first aspect can also consist essentially of 2′-FL,DFL, LNFP-I and LNDFH-I. This HMO mixture preferably has a molar ratioof LNDFH-I relative to (DFL+LNFP-I) of at least 1:10, more preferably atleast 1:7, even more preferably at least 1:5, especially 1:3. This HMOmixture also preferably has a molar ratio of DFL relative to LNFP-I of0.17-6, preferably 0.25-4, more preferably about 1. Also preferably,this mixture has a molar ratio of 2′-FL relative to LNDFH-I of about 1.

The Second Aspect of the Invention

The second aspect is a mixture of HMOs consisting essentially of

-   -   3-FL,    -   a component E which is LNT, LNnT or LNFP-I, and    -   a component F which is        -   LNFP-II when component E is LNT, or        -   LNFP-III when component E is LNnT, or        -   LNDFH-I when component E is LNFP-I.

Preferably, the molar ratio of the component F relative to(3-FL+component E) is at least 1:10, preferably at least 1:7, morepreferably at least 1:5, even more preferably at least 1:3, and/or themolar ratio of 3-FL relative to the component E is 0.05-21, preferably0.13-7.7, more preferably about 1.

The mixture of the second aspect can consist essentially of 3-FL, LNTand LNFP-II. This HMO mixture preferably has a molar ratio of LNFP-IIrelative to (3-FL+LNT) of at least 1:10, more preferably at least 1:7,even more preferably at least 1:5, especially 1:3. This HMO mixture alsopreferably has a molar ratio of 3-FL relative to LNT of 0.05-21,preferably 0.13-7.7, more preferably about 1.

The mixture of the second aspect also can consist essentially of 3-FL,LNnT and LNFP-III. This HMO mixture preferably has a molar ratio ofLNFP-III relative to (3-FL +LNnT) of at least 1:10, more preferably atleast 1:7, even more preferably at least 1:5, especially 1:3. This HMOmixture also preferably has a molar ratio of 3-FL relative to LNnT of0.05-21, preferably 0.13-7.7, more preferably about 1.

The mixture of the second aspect can also consist essentially of 3-FL,LNFP-I and LNDFH-I. This HMO mixture preferably has a molar ratio ofLNDFH-I relative to (3-FL +LNFP-I) of at least 1:10, more preferably atleast 1:7, even more preferably at least 1:5, especially 1:3. This HMOmixture also preferably has a molar ratio of 3-FL relative to LNFP-I of0.05-21, preferably 0.13-7.7, more preferably about 1.

The Third Aspect of the Invention

The third aspect is a process for obtaining a mixture of the firstaspect of the invention, including its preferred and the more preferredembodiments, comprising the steps of reacting a component A and acomponent B in the presence of an α1-3/4 transfucosidase to produce areaction mixture containing the components A, B, C and D, and thenremoving the α1-3/4 transfucosidase from the reaction mixture. Theα1-3/4 transfucosidase can be removed in a conventional manner, e.g., bydenaturing the reaction mixture followed by its centrifugation orultrafiltration.

In carrying out a process of the third aspect this invention, particularrelative concentrations of the component A donor, component B acceptor,the α1-3/4 transfucosidase, the aqueous solvent and the incubationbuffer (e.g. 50 mM Na₃PO₄ or 100 mM KHPO₄) are not critical. In thisregard, the process can be suitably carried out at room temperature(e.g. 15-50, preferably 20-37° C.) at a pH of 6-8, preferably 6.5-7 for15 min to 24 hours.

The Process of the Third Aspect Carried Out with DFL as the Component A

This process involves reacting DFL as a donor and a component B as anacceptor in a molar ratio of 1:5 to 5:1, preferably 1:3 to 3:1, morepreferably 1:2 to 2:1, even more preferably 1:1, in the presence of anα1-3/4 transfucosidase having a conversion rate of at least 10%, up toabout 60%, preferably at least 20%, more preferably at least 30%, forthe reaction of DFL and the component B.

An HMO mixture of this invention can be readily obtained by this processby treating DFL and LNT with an α1-3/4 transfucosidase to produce areaction mixture and then removing the α1-3/4 transfucosidase from thereaction mixture. This process preferably comprises the step ofcontacting DFL and LNT in a molar ratio of preferably 1:5 to 5:1, morepreferably 1:3 to 3:1, even more preferably 1:2 to 2:1, yet even morepreferably 1:1, with an α1-3/4 transfucosidase having a conversion rateof at least 10%, up to about 60%, preferably at least 15%, morepreferably at least 20%, even more preferably at least 25%, yet morepreferably at least 30%, for the reaction of DFL with LNT. The reactionmixture, so-produced, containing LNFP-II, 2′-FL, unreacted DFL, LNT andα1-3/4 transfucosidase, is then subjected to conventional purificationsteps to remove the α1-3/4 transfucosidase. The α1-3/4 transfucosidasecan be removed, e.g., by denaturing it followed by centrifugation orultrafiltration to produce a mixture consisting essentially of DFL, LNT,LNFP-II and 2′-FL.

In the following statements beginning “When the process [. . . ] iscarried out. . . ”, the expression “can be made” is equivalent to theexpression “is obtained”.

When the process of this invention is carried out with a molar ratio ofDFL to LNT of 2:1 to 1:2 and a conversion rate of 20-50%, an HMO mixturehaving a molar ratio of (DFL+LNT) to LNFP-II of 2-13 and a molar ratioof DFL to LNT of 0.33-6 can be made.

When the process is carried out with a molar ratio of DFL to LNT of 1:1and a conversion rate of 20-50%, an HMO mixture having a molar ratio of(DFL+LNT) to LNFP-II of 2-8 and a molar ratio of DFL to LNT of 1 can bemade.

When the process is carried out with a molar ratio of DFL to LNT of 3:1to 1:3 and a conversion rate of 30-50%, an HMO mixture having a molarratio of (DFL+LNT) to LNFP-II of 2-12 and a molar ratio of DFL to LNT of0.2-5 can be made.

When the process is carried out with a molar ratio of DFL to LNT of 2:1to 1:2 and a conversion rate of 30-50%, an HMO mixture having a molarratio of (DFL+LNT) to LNFP-II of 2-8 and a molar ratio of DFL to LNT of0.33-3 can be made.

When the process is carried out with a molar ratio of DFL to LNT of 1:1and a conversion rate of 30-50%, an HMO mixture having a molar ratio of(DFL+LNT) to LNFP-II of 2-4.67 and a molar ratio of DFL to LNT of 1 canbe made.

When the process is carried out with a molar ratio of DFL to LNT of 3:1to 1:3 and a conversion rate of 20-40%, an HMO mixture having a molarratio of (DFL+LNT) to LNFP-II of 3-18 and a molar ratio of DFL to LNT of0.23-4.33 can be made.

When the process is carried out with a molar ratio of DFL to LNT of 2:1to 1:2 and a conversion rate of 20-40%, an HMO mixture having a molarratio of (DFL+LNT) to LNFP-II of 3-13 and a molar ratio of DFL to LNT of0.38-2.67 can be made.

When the process is carried out with a molar ratio of DFL to LNT of 1:1and a conversion rate of 20-40%, an HMO mixture having a molar ratio of(DFL+LNT) to LNFP-II of 3-8 and a molar ratio of DFL to LNT of 1 can bemade.

When the process is carried out with a molar ratio of DFL to LNT of 3:1to 2:1 and a conversion rate of 30-50%, an HMO mixture having a molarratio of (DFL+LNT) to LNFP-II of 4-11.3 and a molar ratio of DFL to LNTof 2.5-5 can be made.

When the process is carried out with a molar ratio of DFL to LNT of 2:1to 1:1 and a conversion rate of 20-40%, an HMO mixture having a molarratio of (DFL+LNT) to LNFP-II of 3-13 and a molar ratio of DFL to LNT of1-2.67 can be made.

When the process is carried out with a molar ratio of DFL to LNT of 1:1to 1:2 and a conversion rate of 20-40%, an HMO mixture having a molarratio of (DFL+LNT) to LNFP-II of 3-13 and a molar ratio of DFL to LNT of0.38-1 can be made.

When the process is carried out with a molar ratio of DFL to LNT of 1:2to 1:3 and a conversion rate of 30-50%, an HMO mixture having a molarratio of (DFL+LNT) to LNFP-II of 4-11.3 and a molar ratio of DFL to LNTof 0.2-0.41 can be made.

An HMO mixture of this invention can also be readily obtained by thisprocess by treating DFL donor and LNnT with an α1-3/4 transfucosidase toproduce a reaction mixture and then removing the α1-3/4 transfucosidasefrom the reaction mixture. This process preferably comprises the step ofcontacting DFL and LNnT in a molar ratio of preferably 1:5to 5:1, morepreferably 1:3 to 3:1, even more preferably 1:2 to 2:1, yet even morepreferably 1:1, with an α1-3/4 transfucosidase having a conversion rateof at least 10%, up to about 60%, preferably at least 15%, morepreferably at least 20%, even more preferably at least 25%, yet morepreferably at least 30%, for the reaction of DFL with LNnT. The reactionmixture, so-produced, containing LNFP-III, 2′-FL, unreacted DFL, LNnTand α1-3/4 transfucosidase, is then subjected to conventionalpurification steps to remove the α1-3/4 transfucosidase. The α1-3/4transfucosidase can be removed, e.g., by denaturing it followed bycentrifugation or ultrafiltration to produce a mixture consistingessentially of DFL, LNnT, LNFP-III and 2′-FL.

In the following statements beginning “When the process [. . . ] iscarried out. . . ”, the expression “can be made” is equivalent to theexpression “is obtained”.

When the process of this invention is carried out with a molar ratio ofDFL to LNnT of 2:1 to 1:2 and a conversion rate of 20-50%, an HMOmixture having a molar ratio of (DFL+LNnT) to LNFP-III of 2-13 and amolar ratio of DFL to LNnT of 0.33-6 can be made.

When the process is carried out with a molar ratio of DFL to LNnT of 1:1and a conversion rate of 20-50%, an HMO mixture having a molar ratio of(DFL+LNnT) to LNFP-III of 2-8 and a molar ratio of DFL to LNnT of 1 canbe made.

When the process is carried out with a molar ratio of DFL to LNnT of 3:1to 1:3 and a conversion rate of 30-50%, an HMO mixture having a molarratio of (DFL+LNnT) to LNFP-III of 2-12 and a molar ratio of DFL to LNnTof 0.2-5 can be made.

When the process is carried out with a molar ratio of DFL to LNnT of 2:1to 1:2 and a conversion rate of 30-50%, an HMO mixture having a molarratio of (DFL+LNnT) to LNFP-III of 2-8 and a molar ratio of DFL to LNnTof 0.33-3 can be made.

When the process is carried out with a molar ratio of DFL to LNnT of 1:1and a conversion rate of 30-50%, an HMO mixture having a molar ratio of(DFL+LNnT) to LNFP-III of 2-4.67 and a molar ratio of DFL to LNnT of 1can be made.

When the process is carried out with a molar ratio of DFL to LNnT of 3:1to 1:3 and a conversion rate of 20-40%, an HMO mixture having a molarratio of (DFL+LNnT) to LNFP-III of 3-18 and a molar ratio of DFL to LNnTof 0.23-4.33 can be made.

When the process is carried out with a molar ratio of DFL to LNnT of 2:1to 1:2 and a conversion rate of 20-40%, an HMO mixture having a molarratio of (DFL+LNnT) to LNFP-III of 3-13 and a molar ratio of DFL to LNnTof 0.38-2.67 can be made.

When the process is carried out with a molar ratio of DFL to LNnT of 1:1and a conversion rate of 20-40%, an HMO mixture having a molar ratio of(DFL+LNnT) to LNFP-III of 3-8 and a molar ratio of DFL to LNnT of 1 canbe made.

When the process is carried out with a molar ratio of DFL to LNnT of 3:1to 2:1 and a conversion rate of 30-50%, an HMO mixture having a molarratio of (DFL+LNnT) to LNFP-III of 4-11.3 and a molar ratio of DFL toLNnT of 2.5-5 can be made.

When the process is carried out with a molar ratio of DFL to LNnT of 2:1to 1:1 and a conversion rate of 20-40%, an HMO mixture having a molarratio of (DFL+LNnT) to LNFP-III of 3-13 and a molar ratio of DFL to LNnTof 1-2.67 can be made.

When the process is carried out with a molar ratio of DFL to LNnT of 1:1to 1:2 and a conversion rate of 20-40%, an HMO mixture having a molarratio of (DFL+LNnT) to LNFP-III of 3-13 and a molar ratio of DFL to LNnTof 0.38-1 can be made.

When the process is carried out with a molar ratio of DFL to LNnT of 1:2to 1:3 and a conversion rate of 30-50%, an HMO mixture having a molarratio of (DFL+LNnT) to LNFP-III of 4-11.3 and a molar ratio of DFL toLNnT of 0.2-0.41 can be made.

An HMO mixture of this invention can also be readily obtained by thisprocess by treating DFL and LNFP-I with an α1-3/4 transfucosidase toproduce a reaction mixture and then removing the α1-3/4 transfucosidasefrom the reaction mixture. This process preferably comprises the step ofcontacting DFL and LNFP-I in a molar ratio of preferably 1:5 to 5:1,more preferably 1:3 to 3:1, even more preferably 1:2 to 2:1, yet evenmore preferably 1:1, with an α1-3/4 transfucosidase having a conversionrate of at least 10%, up to about 60%, preferably at least 15%, morepreferably at least 20%, even more preferably at least 25%, yet morepreferably at least 30%, for the reaction of DFL with LNFP-I. Thereaction mixture, so-produced, containing LNDFH-I, 2′-FL, unreacted DFL,LNFP-I and α1-3/4 transfucosidase, is then subjected to conventionalpurification steps to remove the α1-3/4 transfucosidase. The α1-3/4transfucosidase can be removed, e.g., by denaturing it followed bycentrifugation or ultrafiltration to produce a mixture consistingessentially of DFL, LNFP-I, LNDFH-I and 2′-FL.

In the following statements beginning “When the process [. . . ] iscarried out. . . ”, the expression “can be made” is equivalent to theexpression “is obtained”.

When the process of this invention is carried out with a molar ratio ofDFL to LNFP-I of 2:1 to 1:2 and a conversion rate of 20-50%, an HMOmixture having a molar ratio of (DFL+LNFP-I) to LNDFH-I of 2-13 and amolar ratio of DFL to LNFP-I of 0.33-6 can be made.

When the process is carried out with a molar ratio of DFL to LNFP-I of1:1 and a conversion rate of 20-50%, an HMO mixture having a molar ratioof (DFL+LNFP-I) to LNDFH-I of 2-8 and a molar ratio of DFL to LNFP-I of1 can be made.

When the process is carried out with a molar ratio of DFL to LNFP-I of3:1 to 1:3 and a conversion rate of 30-50%, an HMO mixture having amolar ratio of (DFL+LNFP-I) to LNDFH-I of 2-12 and a molar ratio of DFLto LNFP-I of 0.2-5 can be made.

When the process is carried out with a molar ratio of DFL to LNFP-I of2:1 to 1:2 and a conversion rate of 30-50%, an HMO mixture having amolar ratio of (DFL+LNFP-I) to LNDFH-I of 2-8 and a molar ratio of DFLto LNFP-I of 0.33-3 can be made.

When the process is carried out with a molar ratio of DFL to LNFP-I of1:1 and a conversion rate of 30-50%, an HMO mixture having a molar ratioof (DFL+LNFP-I) to LNDFH-I of 2-4.67 and a molar ratio of DFL to LNFP-Iof 1 can be made.

When the process is carried out with a molar ratio of DFL to LNFP-I of3:1 to 1:3 and a conversion rate of 20-40%, an HMO mixture having amolar ratio of (DFL+LNFP-I) to LNDFH-I of 3-18 and a molar ratio of DFLto LNFP-I of 0.23-4.33 can be made.

When the process is carried out with a molar ratio of DFL to LNFP-I of2:1 to 1:2 and a conversion rate of 20-40%, an HMO mixture having amolar ratio of (DFL+LNFP-I) to LNDFH-I of 3-13 and a molar ratio of DFLto LNFP-I of 0.38-2.67 can be made.

When the process is carried out with a molar ratio of DFL to LNFP-I of1:1 and a conversion rate of 20-40%, an HMO mixture having a molar ratioof (DFL+LNFP-I) to LNDFH-I of 3-8 and a molar ratio of DFL to LNFP-I of1 can be made.

When the process is carried out with a molar ratio of DFL to LNFP-I of3:1 to 2:1 and a conversion rate of 30-50%, an HMO mixture having amolar ratio of (DFL+LNFP-I) to LNDFH-I of 4-11.3 and a molar ratio ofDFL to LNFP-I of 2.5-5 can be made.

When the process is carried out with a molar ratio of DFL to LNFP-I of2:1 to 1:1 and a conversion rate of 20-40%, an HMO mixture having amolar ratio of (DFL+LNFP-I) to LNDFH-I of 3-13 and a molar ratio of DFLto LNFP-I of 1-2.67 can be made.

When the process is carried out with a molar ratio of DFL to LNFP-I of1:1 to 1:2 and a conversion rate of 20-40%, an HMO mixture having amolar ratio of (DFL+LNFP-I) to LNDFH-I of 3-13 and a molar ratio of DFLto LNFP-I of 0.38-1 can be made.

When the process is carried out with a molar ratio of DFL to LNFP-I of1:2 to 1:3 and a conversion rate of 30-50%, an HMO mixture having amolar ratio of (DFL+LNFP-I) to LNDFH-I of 4-11.3 and a molar ratio ofDFL to LNFP-I of 0.2-0.41 can be made.

The Process of the Third Aspect, Carried Out with 3-FL as the ComponentA

Alternatively, the process of the third aspect can be carried out with3-FL as a donor and a component B as an acceptor in a molar ratio of 1:5to 5:1, preferably 1:3 to 3:1, more preferably 1:2 to 2:1, even morepreferably 1:1, in the presence of an α1-3/4 transfucosidase having aconversion rate of at least 35%, up to about 80%, preferably at least40%, more preferably at least 50%, for the reaction of 3-FL and thecomponent B. The reaction mixture, so-produced can then be subjected toconventional purification steps to remove the α1-3/4 transfucosidase.The α1-3/4 transfucosidase can be removed, e.g., by denaturing itfollowed by centrifugation or ultrafiltration.

In a preferred embodiment of the alternative process of the thirdaspect, an HMO mixture of this invention can be readily obtained bytreating 3-FL and LNFP-I or 2′-FL with an α1-3/4 transfucosidase toproduce a reaction mixture and then removing the α1-3/4 transfucosidasefrom the reaction mixture. This process preferably comprises the step ofcontacting 3-FL and LNFP-I or2′-FL in a molar ratio of 1:3 to 3:1,preferably 1:2 to 2:1, more preferably 1:1, in the presence of an α1-3/4transfucosidase having a conversion rate of at least 35%, up to about70%, preferably at least 40%, more preferably at least 50%, for thereaction of 3-FL with LNFP-I or 2′-FL.

In a more preferred embodiment, an HMO mixture consisting essentially of3-FL, LNFP-I, LNDFH-I and lactose can be readily obtained by treating3-FL and LNFP-I in a molar ratio of preferably 1:5 to 5:1, morepreferably 1:3 to 3:1, even more preferably 1:2 to 2:1, yet even morepreferably 1:1, with an α1-3/4 transfucosidase having a conversion rateof at least 35%, up to about 70%, preferably at least 40%, morepreferably at least 50%, for the reaction of 3-FL with LNFP-I.

In another more preferred embodiment, an HMO mixture consistingessentially of 3-FL, 2′-FL, DFL and lactose can be readily obtained bytreating 3-FL and 2′-FL in a molar ratio of preferably 1:5 to 5:1, morepreferably 1:3 to 3:1, even more preferably 1:2 to 2:1, yet even morepreferably 1:1 with an α1-3/4 transfucosidase having a conversion rateof at least 10%, up to about 70%, preferably at least 20%, morepreferably at least 35%, even more preferably at least 40%, yet morepreferably at least 50%, for the reaction of 3-FL with 2′-FL.

In another preferred embodiment of the alternative process of the thirdaspect, an HMO mixture of this invention can be readily obtained bytreating 3-FL and LNT or LNnT with an α1-3/4 transfucosidase to producea reaction mixture and then the α1-3/4 transfucosidase is removed fromthe reaction mixture. This process preferably involves reacting 3-FL andLNT or LNnT in a molar ratio of 1:3 to 3:1, preferably 1:2 to 2:1, morepreferably 1:1, in the presence of an α1-3/4 transfucosidase having aconversion rate of at least 35%, up to about 70%, preferably at least40%, more preferably at least 50%, for the reaction of 3-FL with LNT orLNnT.

In a more preferred embodiment, an HMO mixture consisting essentially of3-FL, LNT, LNFP-II and lactose, and in which:

-   -   the molar ratio of (3-FL+LNT) relative to LNFP-II is 0.8-9.5,        and    -   the molar ratio of lactose relative to LNFP-II is about 1,        provided that one of the molar ratios 3-FL to LNFP-II and LNT to        LNFP-II is not more than 2, can be readily obtained by treating        3-FL and LNT in a molar ratio of preferably 1:5 to 5:1, more        preferably 1:3 to 3:1, even more preferably 1:2 to 2:1, yet even        more preferably 1:1, and with an α1-3/4 transfucosidase having a        conversion rate of at least 35%, up to about 70%, preferably at        least 40%, more preferably at least 50% for the reaction of 3-FL        with LNT.

In another more preferred embodiment, an HMO mixture consistingessentially of 3-FL, LNnT, LNFP-III and lactose, and in which:

-   -   the molar ratio of (3-FL+LNnT) relative to LNFP-III is 0.8-9.5,        and    -   the molar ratio of lactose relative to LNFP-III is about 1,        provided that one of the molar ratios 3-FL to LNFP-III and LNnT        to LNFP-III is not more than 2, can be obtained by treating 3-FL        and LNnT in a molar ratio of preferably 1:5 to 5:1, more        preferably 1:3 to 3:1, even more preferably 1:2 to 2:1, yet even        more preferably 1:1, with an α1-3/4 transfucosidase having a        conversion rate of at least 35%, up to about 70%, preferably at        least 40%, more preferably at least 50% for the reaction of 3-FL        with LNnT.

The Fourth Aspect of the Invention

The fourth aspect is a process for obtaining a mixture of HMOsconsisting essentially of

-   -   3-FL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL, and    -   a component C, which is:        -   LNFP-II when component B is LNT, or        -   LNFP-III when component B is LNnT, or        -   LNDFH-I when component B is LNFP-I, or        -   DFL when component B is 2′-FL,            preferably a mixture of the second aspect, including its            preferred and the more preferred embodiments, comprising the            steps of reacting 3-FL as a donor and a component B as an            acceptor in the presence of an α1-3/4 transfucosidase to            produce a reaction mixture, and then removing lactose and            the α1-3/4 transfucosidase from the reaction mixture. The            α1-3/4 transfucosidase can be removed, e.g. by denaturing it            followed by centrifugation or ultrafiltration. The lactose            can be separated from the 3-FL, component B and component C,            e.g. by cascade ultra- and/or nanofiltration, or the lactose            can first be treated with lactase to degrade it to glucose            and galactose which can then be separated from the 3-FL,            component B and component C by ultra- and/or nanofiltration.

In carrying out a process of the fourth aspect of this invention,particular relative concentrations of the 3-FL donor, component Bacceptor, the α1-3/4 transfucosidase, the aqueous solvent and theincubation buffer (e.g. 50 mM Na₃PO₄ or 100 mM KHPO₄) are not critical.In this regard, the process can be suitably carried out at roomtemperature (e.g. 15-50, preferably 20-37° C.) at a pH of 6-8,preferably 6.5-7 for 15 min to 24 hours.

The process preferably comprises the step of reacting 3-FL and thecomponent B in a molar ratio of 1:5 to 5:1, preferably 1:3 to 3:1, morepreferably 1:2 to 2:1, even more preferably 1:1, in the presence of anα1-3/4 transfucosidase having a conversion rate of at least 35%, up toabout 80%, preferably at least 40%, more preferably at least 50%, forthe reaction of 3-FL and a component B.

In a preferred embodiment of the process of the fourth aspect, an HMOmixture consisting essentially of 3-FL, LNT and LNFP-II can be readilyobtained by treating 3-FL and LNT with an α1-3/4 transfucosidase toproduce a reaction mixture and then removing lactose and the α1-3/4transfucosidase from the reaction mixture as described above. Thisprocess preferably comprises the step of contacting 3-FL and LNT in amolar ratio of preferably 1:5 to 5:1, more preferably 1:3 to 3:1, evenmore preferably 1:2 to 2:1, yet even more preferably 1:1, with an α1-3/4transfucosidase having a conversion rate of at least 10%, up to about70%, preferably at least 20%, more preferably at least 35%, even morepreferably at least 40%, yet more preferably at least 50%, for thereaction of 3-FL and LNT. From the reaction mixture, so-produced,containing LNFP-II, lactose, unreacted 3-FL, LNT and the α1-3/4transfucosidase, the α1-3/4 transfucosidase and the lactose can then beremoved as described above.

In another preferred embodiment of the process of the fourth aspect, anHMO mixture consisting essentially of 3-FL, LNnT and LNFP-III can bereadily obtained by treating 3-FL and LNnT with an α1-3/4transfucosidase to produce a reaction mixture and then removing lactoseand the α1-3/4 transfucosidase from the reaction mixture as describedabove. This process preferably comprises the step of contacting 3-FL andLNnT in a molar ratio of 1:5 to 5:1, more preferably 1:3 to 3:1, evenmore preferably 1:2 to 2:1, yet even more preferably 1:1, with an α1-3/4transfucosidase having a conversion rate of at least 10%, up to about70%, preferably at least 20%, more preferably at least 35%, even morepreferably at least 40%, yet more preferably at least 50%, for thereaction of 3-FL and LNnT. From the reaction mixture, so-produced,containing LNFP-III, lactose, unreacted 3-FL, LNnT and the α1-3/4transfucosidase, α1-3/4 transfucosidase and the lactose can then beremoved as described above.

In yet another preferred embodiment of the process of the fourth aspect,an HMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-I can bereadily obtained by treating 3-FL and LNFP-I with an α1-3/4transfucosidase to produce a reaction mixture and then removing lactoseand the α1-3/4 transfucosidase from the reaction mixture as describedabove. This process preferably comprises the step of contacting 3-FL andLNFP-I in a molar ratio of preferably 1:5 to 5:1, more preferably 1:3 to3:1, even more preferably 1:2 to 2:1, yet even more preferably 1:1, withan α1-3/4 transfucosidase having a conversion rate of at least 10%, upto about 70%, preferably at least 20%, more preferably at least 35%,even more preferably at least 40%, yet more preferably at least 50%, forthe reaction of 3-FL and LNFP-I. From the reaction mixture, so-produced,containing LNDFH-I, lactose, unreacted 3-FL, LNFP-I and α1-3/4transfucosidase, the α1-3/4 transfucosidase and the lactose can then beremoved as described above.

In a further preferred embodiment of the process of the fourth aspect,an HMO mixture consisting essentially of 3-FL, 2′-FL and DFL can bereadily obtained by treating 3-FL and 2′-FL acceptor, with an α1-3/4transfucosidase to produce a reaction mixture containing DFL, lactose,unreacted 3-FL, 2′-FL and α1-3/4 transfucosidase. The mixture is then besubjected to conventional purification steps to remove the lactose andthe α1-3/4 transfucosidase as described above. This HMO mixtureso-obtained preferably has a molar ratio of DFL relative to (3-FL+2′-FL)of at least 1:10, more preferably at least 1:7, even more preferably atleast 1:5, especially 1:3. This HMO mixture also has a molar ratio of3-FL relative to 2′-FL of preferably 0.05-21, more preferably 0.13-7.7,even more preferably about 1.

Particularly Preferred Third and Fourth Aspects of the Invention

In the following statements beginning “When the process [. . . ] iscarried out . . . ”, the expression “can be made” is equivalent to theexpression “is obtained”.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 2:1 to 1:2 and a conversion rate of35-70%, an HMO mixture consisting essentially of 3-FL, LNT, LNFP-II andlactose, or an HMO mixture consisting essentially of 3-FL, LNT andLNFP-II, respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of0.8-6.6 and a molar ratio of 3-FL to LNT of 0.2-4.5 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 1:1 and a conversion rate of 35-70%, anHMO mixture consisting essentially of 3-FL, LNT, LNFP-II and lactose, oran HMO mixture consisting essentially of 3-FL, LNT and LNFP-II,respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of 0.8-3.8and a molar ratio of 3-FL to LNT of 1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 3:1 to 1:3 and a conversion rate of40-70%, an HMO mixture consisting essentially of 3-FL, LNT, LNFP-II andlactose, or an HMO mixture consisting essentially of 3-FL, LNT andLNFP-II, respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of0.8-8 and a molar ratio of 3-FL to LNT of 0.13-7.7 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 2:1 to 1:2 and a conversion rate of40-70%, an HMO mixture consisting essentially of 3-FL, LNT, LNFP-II andlactose, or an HMO mixture consisting essentially of 3-FL, LNT andLNFP-II, respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of0.8-5.5 and a molar ratio of 3-FL to LNT of 0.2-4.5 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 1:1 and a conversion rate of 40-70%, anHMO mixture consisting essentially of 3-FL, LNT, LNFP-II and lactose, oran HMO mixture consisting essentially of 3-FL, LNT and LNFP-II,respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of 0.8-3 anda molar ratio of 3-FL to LNT of 1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 3:1 to 1:3 and a conversion rate of50-70%, an HMO mixture consisting essentially of 3-FL, LNT, LNFP-II andlactose, or an HMO mixture consisting essentially of 3-FL, LNT andLNFP-II, respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of0.8-6 and a molar ratio of 3-FL to LNT of 0.13-7.7 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 2:1 to 1:2 and a conversion rate of50-70%, an HMO mixture consisting essentially of 3-FL, LNT, LNFP-II andlactose, or an HMO mixture consisting essentially of 3-FL, LNT andLNFP-II, respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of0.8-4 and a molar ratio of 3-FL to LNT of 0.2-4.5 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 1:1 and a conversion rate of 50-70%, anHMO mixture consisting essentially of 3-FL, LNT, LNFP-II and lactose, oran HMO mixture consisting essentially of 3-FL, LNT and LNFP-II,respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of 0.8-2 anda molar ratio of 3-FL to LNT of 1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 3:1 to 1:3 and a conversion rate of40-60%, an HMO mixture consisting essentially of 3-FL, LNT, LNFP-II andlactose, or an HMO mixture consisting essentially of 3-FL, LNT andLNFP-II, respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of1.3-8 and a molar ratio of 3-FL to LNT of 0.17-6 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 2:1 to 1:2 and a conversion rate of40-60%, an HMO mixture consisting essentially of 3-FL, LNT, LNFP-II andlactose, or an HMO mixture consisting essentially of 3-FL, LNT andLNFP-II, respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of2-6.6 and a molar ratio of 3-FL to LNT of 0.25-3.5 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 1:1 and a conversion rate of 40-60%, anHMO mixture consisting essentially of 3-FL, LNT, LNFP-II and lactose, oran HMO mixture consisting essentially of 3-FL, LNT and LNFP-II,respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of 1.3-3 anda molar ratio of 3-FL to LNT of 1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 5:1 to 3:1 and a conversion rate of50-80%, an HMO mixture consisting essentially of 3-FL, LNT, LNFP-II andlactose, or an HMO mixture consisting essentially of 3-FL, LNT andLNFP-II, respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of3-10 and a molar ratio of 3-FL to LNT of 5-21 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 3:1 to 2:1 and a conversion rate of40-70%, an HMO mixture consisting essentially of 3-FL, LNT, LNFP-II andlactose, or an HMO mixture consisting essentially of 3-FL, LNT andLNFP-II, respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of2-8 and a molar ratio of 3-FL to LNT of 2.5-7.7 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 2:1 to 1:1 and a conversion rate of35-55%, an HMO mixture consisting essentially of 3-FL, LNT, LNFP-II andlactose, or an HMO mixture consisting essentially of 3-FL, LNT andLNFP-II, respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of1.6-6.6 and a molar ratio of 3-FL to LNT of 1-3.3 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 1:1 to 1:2 and a conversion rate of35-55%, an HMO mixture consisting essentially of 3-FL, LNT, LNFP-II andlactose, or an HMO mixture consisting essentially of 3-FL, LNT andLNFP-II, respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of1.6-6.6 and a molar ratio of 3-FL to LNT of 0.3-1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 1:2 to 1:3 and a conversion rate of40-70%, an HMO mixture consisting essentially of 3-FL, LNT, LNFP-II andlactose, or an HMO mixture consisting essentially of 3-FL, LNT andLNFP-II, respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of2-8 and a molar ratio of 3-FL to LNT of 0.13-0.4 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNT of 1:3 to 1:5 and a conversion rate of50-80%, an HMO mixture consisting essentially of 3-FL, LNT, LNFP-II andlactose, or an HMO mixture consisting essentially of 3-FL, LNT andLNFP-II, respectively, having a molar ratio of (3-FL+LNT) to LNFP-II of3-10 and a molar ratio of 3-FL to LNT of 0.05-0.2 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 2:1 to 1:2 and a conversion rate of35-70%, an HMO mixture consisting essentially of 3-FL, LNnT, LNFP-IIIand lactose, or an HMO mixture consisting essentially of 3-FL, LNnT andLNFP-III, respectively, having a molar ratio of (3-FL+LNnT) to LNFP-IIIof 0.8-6.6 and a molar ratio of 3-FL to LNnT of 0.2-4.5 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 1:1 and a conversion rate of 35-70%, anHMO mixture consisting essentially of 3-FL, LNnT, LNFP-III and lactose,or an HMO mixture consisting essentially of 3-FL, LNnT and LNFP-III,respectively, having a molar ratio of (3-FL+LNnT) to LNFP-III of 0.8-3.8and a molar ratio of 3-FL to LNnT of 1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 3:1 to 1:3 and a conversion rate of40-70%, an HMO mixture consisting essentially of 3-FL, LNnT, LNFP-IIIand lactose, or an HMO mixture consisting essentially of 3-FL, LNnT andLNFP-III, respectively, having a molar ratio of (3-FL+LNnT) to LNFP-IIIof 0.8-8 and a molar ratio of 3-FL to LNnT of 0.13-7.7 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 2:1 to 1:2 and a conversion rate of40-70%, an HMO mixture consisting essentially of 3-FL, LNnT, LNFP-IIIand lactose, or an HMO mixture consisting essentially of 3-FL, LNnT andLNFP-III, respectively, having a molar ratio of (3-FL+LNnT) to LNFP-IIIof 0.8-5.5 and a molar ratio of 3-FL to LNnT of 0.2-4.5 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 1:1 and a conversion rate of 40-70%, anHMO mixture consisting essentially of 3-FL, LNnT, LNFP-III and lactose,or an HMO mixture consisting essentially of 3-FL, LNnT and LNFP-III,respectively, having a molar ratio of (3-FL+LNnT) to LNFP-III of 0.8-3and a molar ratio of 3-FL to LNnT of 1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 3:1 to 1:3 and a conversion rate of50-70%, an HMO mixture consisting essentially of 3-FL, LNnT, LNFP-IIIand lactose, or an HMO mixture consisting essentially of 3-FL, LNnT andLNFP-III, respectively, having a molar ratio of (3-FL+LNnT) to LNFP-IIIof 0.8-6 and a molar ratio of 3-FL to LNnT of 0.13-7.7 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 2:1 to 1:2 and a conversion rate of50-70%, an HMO mixture consisting essentially of 3-FL, LNnT, LNFP-IIIand lactose, or an HMO mixture consisting essentially of 3-FL, LNnT andLNFP-III, respectively, having a molar ratio of (3-FL+LNnT) to LNFP-IIIof 0.8-4 and a molar ratio of 3-FL to LNnT of 0.2-4.5 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 1:1 and a conversion rate of 50-70%, anHMO mixture consisting essentially of 3-FL, LNnT, LNFP-III and lactose,or an HMO mixture consisting essentially of 3-FL, LNnT and LNFP-III,respectively, having a molar ratio of (3-FL+LNnT) to LNFP-III of 0.8-2and a molar ratio of 3-FL to LNnT of 1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 3:1 to 1:3 and a conversion rate of40-60%, an HMO mixture consisting essentially of 3-FL, LNnT, LNFP-IIIand lactose, or an HMO mixture consisting essentially of 3-FL, LNnT andLNFP-III, respectively, having a molar ratio of (3-FL+LNnT) to LNFP-IIIof 1.3-8 and a molar ratio of 3-FL to LNnT of 0.17-6 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 2:1 to 1:2 and a conversion rate of40-60%, an HMO mixture consisting essentially of 3-FL, LNnT, LNFP-IIIand lactose, or an HMO mixture consisting essentially of 3-FL, LNnT andLNFP-III, respectively, having a molar ratio of (3-FL+LNnT) to LNFP-IIIof 2-6.6and a molar ratio of 3-FL to LNnT of 0.25-3.5 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 1:1 and a conversion rate of 40-60%, anHMO mixture consisting essentially of 3-FL, LNnT, LNFP-III and lactose,or an HMO mixture consisting essentially of 3-FL, LNnT and LNFP-III,respectively, having a molar ratio of (3-FL+LNnT) to LNFP-III of 1.3-3and a molar ratio of 3-FL to LNnT of 1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 5:1 to 3:1 and a conversion rate of50-80%, an HMO mixture consisting essentially of 3-FL, LNnT, LNFP-IIIand lactose, or an HMO mixture consisting essentially of 3-FL, LNnT andLNFP-III, respectively, having a molar ratio of (3-FL+LNnT) to LNFP-IIIof 3-10 and a molar ratio of 3-FL to LNnT of 5-21 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 3:1 to 2:1 and a conversion rate of40-70%, an HMO mixture consisting essentially of 3-FL, LNnT, LNFP-IIIand lactose, or an HMO mixture consisting essentially of 3-FL, LNnT andLNFP-III, respectively, having a molar ratio of (3-FL+LNnT) to LNFP-IIIof 2-8and a molar ratio of 3-FL to LNnT of 2.5-7.7 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 2:1 to 1:1 and a conversion rate of35-55%, an HMO mixture consisting essentially of 3-FL, LNnT, LNFP-IIIand lactose, or an HMO mixture consisting essentially of 3-FL, LNnT andLNFP-III, respectively, having a molar ratio of (3-FL+LNnT) to LNFP-IIIof 1.6-6.6 and a molar ratio of 3-FL to LNnT of 1-3.3 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 1:1 to 1:2 and a conversion rate of35-55%, an HMO mixture consisting essentially of 3-FL, LNnT, LNFP-IIIand lactose, or an HMO mixture consisting essentially of 3-FL, LNnT andLNFP-III, respectively, having a molar ratio of (3-FL+LNnT) to LNFP-IIIof 1.6-6.6 and a molar ratio of 3-FL to LNnT of 0.3-1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 1:2 to 1:3 and a conversion rate of40-70%, an HMO mixture consisting essentially of 3-FL, LNnT, LNFP-IIIand lactose, or an HMO mixture consisting essentially of 3-FL, LNnT andLNFP-III, respectively, having a molar ratio of (3-FL+LNnT) to LNFP-IIIof 2-8 and a molar ratio of 3-FL to LNnT of 0.13-0.4 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNnT of 1:3 to 1:5 and a conversion rate of50-80%, an HMO mixture consisting essentially of 3-FL, LNnT, LNFP-IIIand lactose, or an HMO mixture consisting essentially of 3-FL, LNnT andLNFP-III, respectively, having a molar ratio of (3-FL+LNnT) to LNFP-IIIof 3-10 and a molar ratio of 3-FL to LNnT of 0.05-0.2 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 2:1 to 1:2 and a conversion rate of35-70%, an HMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-Iand lactose, or an HMO mixture consisting essentially of 3-FL, LNFP-Iand LNDFH-I, respectively, having a molar ratio of (3-FL+LNFP-I) toLNDFH-I of 0.8-6.6 and a molar ratio of 3-FL to LNFP-I of 0.2-4.5 can bemade.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 1:1 and a conversion rate of 35-70%, anHMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-I and lactose,or an HMO mixture consisting essentially of 3-FL, LNFP-I and LNDFH-I,respectively, having a molar ratio of (3-FL+LNFP-I) to LNDFH-I of0.8-3.8 and a molar ratio of 3-FL to LNFP-I of 1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 3:1 to 1:3 and a conversion rate of40-70%, an HMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-Iand lactose, or an HMO mixture consisting essentially of 3-FL, LNFP-Iand LNDFH-I, respectively, having a molar ratio of (3-FL+LNFP-I) toLNDFH-I of 0.8-8 and a molar ratio of 3-FL to LNFP-I of 0.13-7.7 can bemade.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 2:1 to 1:2 and a conversion rate of40-70%, an HMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-Iand lactose, or an HMO mixture consisting essentially of 3-FL, LNFP-Iand LNDFH-I, respectively, having a molar ratio of (3-FL+LNFP-I) toLNDFH-I of 0.8-5.5 and a molar ratio of 3-FL to LNFP-I of 0.2-4.5 can bemade.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 1:1 and a conversion rate of 40-70%, anHMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-I and lactose,or an HMO mixture consisting essentially of 3-FL, LNFP-I and LNDFH-I,respectively, having a molar ratio of (3-FL+LNFP-I) to LNDFH-I of 0.8-3and a molar ratio of 3-FL to LNFP-I of 1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 3:1 to 1:3 and a conversion rate of50-70%, an HMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-Iand lactose, or an HMO mixture consisting essentially of 3-FL, LNFP-Iand LNDFH-I, respectively, having a molar ratio of (3-FL+LNFP-I) toLNDFH-I of 0.8-6 and a molar ratio of 3-FL to LNFP-I of 0.13-7.7 can bemade.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 2:1 to 1:2 and a conversion rate of50-70%, an HMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-Iand lactose, or an HMO mixture consisting essentially of 3-FL, LNFP-Iand LNDFH-I, respectively, having a molar ratio of (3-FL+LNFP-I) toLNDFH-I of 0.8-4 and a molar ratio of 3-FL to LNFP-I of 0.2-4.5 can bemade.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 1:1 and a conversion rate of 50-70%, anHMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-I and lactose,or an HMO mixture consisting essentially of 3-FL, LNFP-I and LNDFH-I,respectively, having a molar ratio of (3-FL+LNFP-I) to LNDFH-I of 0.8-2and a molar ratio of 3-FL to LNFP-I of 1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 3:1 to 1:3 and a conversion rate of40-60%, an HMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-Iand lactose, or an HMO mixture consisting essentially of 3-FL, LNFP-Iand LNDFH-I, respectively, having a molar ratio of (3-FL+LNFP-I) toLNDFH-I of 1.3-8 and a molar ratio of 3-FL to LNFP-I of 0.17-6 can bemade.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 2:1 to 1:2 and a conversion rate of40-60%, an HMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-Iand lactose, or an HMO mixture consisting essentially of 3-FL, LNFP-Iand LNDFH-I, respectively, having a molar ratio of (3-FL+LNFP-I) toLNDFH-I of 2-6.6 and a molar ratio of 3-FL to LNFP-I of 0.25-3.5 can bemade.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 1:1 and a conversion rate of 40-60%, anHMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-I and lactose,or an HMO mixture consisting essentially of 3-FL, LNFP-I and LNDFH-I,respectively, having a molar ratio of (3-FL+LNFP-I) to LNDFH-I of 1.3-3and a molar ratio of 3-FL to LNFP-I of 1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 5:1 to 3:1 and a conversion rate of50-80%, an HMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-Iand lactose, or an HMO mixture consisting essentially of 3-FL, LNFP-Iand LNDFH-I, respectively, having a molar ratio of (3-FL+LNFP-I) toLNDFH-I of 3-10 and a molar ratio of 3-FL to LNFP-I of 5-21 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 3:1 to 2:1 and a conversion rate of40-70%, an HMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-Iand lactose, or an HMO mixture consisting essentially of 3-FL, LNFP-Iand LNDFH-I, respectively, having a molar ratio of (3-FL+LNFP-I) toLNDFH-I of 2-8 and a molar ratio of 3-FL to LNFP-I of 2.5-7.7 can bemade.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 2:1 to 1:1 and a conversion rate of35-55%, an HMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-Iand lactose, or an HMO mixture consisting essentially of 3-FL, LNFP-Iand LNDFH-I, respectively, having a molar ratio of (3-FL+LNFP-I) toLNDFH-I of 1.6-6.6 and a molar ratio of 3-FL to LNFP-I of 1-3.3 can bemade.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 1:1 to 1:2 and a conversion rate of35-55%, an HMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-Iand lactose, or an HMO mixture consisting essentially of 3-FL, LNFP-Iand LNDFH-I, respectively, having a molar ratio of (3-FL+LNFP-I) toLNDFH-I of 1.6-6.6 and a molar ratio of 3-FL to LNFP-I of 0.3-1 can bemade.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 1:2 to 1:3 and a conversion rate of40-70%, an HMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-Iand lactose, or an HMO mixture consisting essentially of 3-FL, LNFP-Iand LNDFH-I, respectively, having a molar ratio of (3-FL+LNFP-I) toLNDFH-I of 2-8 and a molar ratio of 3-FL to LNFP-I of 0.13-0.4 can bemade.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to LNFP-I of 1:3 to 1:5 and a conversion rate of50-80%, an HMO mixture consisting essentially of 3-FL, LNFP-I, LNDFH-Iand lactose, or an HMO mixture consisting essentially of 3-FL, LNFP-Iand LNDFH-I, respectively, having a molar ratio of (3-FL+LNFP-I) toLNDFH-I of 3-10 and a molar ratio of 3-FL to LNFP-I of 0.05-0.2 can bemade.

An HMO mixture consisting essentially of 3-FL, 2′-FL, DFL and lactose orconsisting essentially of 3-FL, 2′-FL and DFL can be obtained bycarrying out a process of the third or the fourth aspect, respectively,with 3-FL and 2′-FL in a molar ratio of preferably 1:5 to 5:1, morepreferably 1:3 to 3:1, even more preferably 1:2 to 2:1, yet even morepreferably 1:1, and with an α1-3/4 transfucosidase having a conversionrate of at least 35%, up to about 70%, preferably at least 40%, morepreferably at least 50%.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 2:1 to 1:2 and a conversion rate of35-70%, an HMO mixture consisting essentially of 3-FL, 2′-FL, DFL andlactose, or an HMO mixture consisting essentially of 3-FL, 2′-FL andDFL, respectively, having a molar ratio of (3-FL+2′-FL) to DFL of0.8-6.6 and a molar ratio of 3-FL to 2′-FL of 0.2-4.5 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 1:1 and a conversion rate of 35-70%, anHMO mixture consisting essentially of 3-FL, 2′-FL, DFL and lactose, oran HMO mixture consisting essentially of 3-FL, 2′-FL and DFL,respectively, having a molar ratio of (3-FL+2′-FL) to DFL of 0.8-3.8 anda molar ratio of 3-FL to 2′-FL of 1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 3:1 to 1:3 and a conversion rate of40-70%, an HMO mixture consisting essentially of 3-FL, 2′-FL, DFL andlactose, or an HMO mixture consisting essentially of 3-FL, 2′-FL andDFL, respectively, having a molar ratio of (3-FL+2′-FL) to DFL of 0.8-8and a molar ratio of 3-FL to 2′-FL of 0.13-7.7 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 2:1 to 1:2 and a conversion rate of40-70%, an HMO mixture consisting essentially of 3-FL, 2′-FL, DFL andlactose, or an HMO mixture consisting essentially of 3-FL, 2′-FL andDFL, respectively, having a molar ratio of (3-FL+2′-FL) to DFL of0.8-5.5 and a molar ratio of 3-FL to 2′-FL of 0.2-4.5 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 1:1 and a conversion rate of 40-70%, anHMO mixture consisting essentially of 3-FL, 2′-FL, DFL and lactose, oran HMO mixture consisting essentially of 3-FL, 2′-FL and DFL,respectively, having a molar ratio of (3-FL+2′-FL) to DFL of 0.8-3 and amolar ratio of 3-FL to 2′-FL of 1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 3:1 to 1:3 and a conversion rate of50-70%, an HMO mixture consisting essentially of 3-FL, 2′-FL, DFL andlactose, or an HMO mixture consisting essentially of 3-FL, 2′-FL andDFL, respectively, having a molar ratio of (3-FL+2′-FL) to DFL of 0.8-6and a molar ratio of 3-FL to 2′-FL of 0.13-7.7 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 2:1 to 1:2 and a conversion rate of50-70%, an HMO mixture consisting essentially of 3-FL, 2′-FL, DFL andlactose, or an HMO mixture consisting essentially of 3-FL, 2′-FL andDFL, respectively, having a molar ratio of (3-FL+2′-FL) to DFL of 0.8-4and a molar ratio of 3-FL to 2′-FL of 0.2-4.5 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 1:1 and a conversion rate of 50-70%, anHMO mixture consisting essentially of 3-FL, 2′-FL, DFL and lactose, oran HMO mixture consisting essentially of 3-FL, 2′-FL and DFL,respectively, having a molar ratio of (3-FL+2′-FL) to DFL of 0.8-2 and amolar ratio of 3-FL to 2′-FL of 1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 3:1 to 1:3 and a conversion rate of40-60%, an HMO mixture consisting essentially of 3-FL, 2′-FL, DFL andlactose, or an HMO mixture consisting essentially of 3-FL, 2′-FL andDFL, respectively, having a molar ratio of (3-FL+2′-FL) to DFL of 1.3-8and a molar ratio of 3-FL to 2′-FL of 0.17-6 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 2:1 to 1:2 and a conversion rate of40-60%, an HMO mixture consisting essentially of 3-FL, 2′-FL, DFL andlactose, or an HMO mixture consisting essentially of 3-FL, 2′-FL andDFL, respectively, having a molar ratio of (3-FL+2′-FL) to DFL of 2-6.6and a molar ratio of 3-FL to 2′-FL of 0.25-3.5 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 1:1 and a conversion rate of 40-60%, anHMO mixture consisting essentially of 3-FL, 2′-FL, DFL and lactose, oran HMO mixture consisting essentially of 3-FL, 2′-FL and DFL,respectively, having a molar ratio of (3-FL+2′-FL) to DFL of 1.3-3 and amolar ratio of 3-FL to 2′-FL of 1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 5:1 to 3:1 and a conversion rate of50-80%, an HMO mixture consisting essentially of 3-FL, 2′-FL, DFL andlactose, or an HMO mixture consisting essentially of 3-FL, 2′-FL andDFL, respectively, having a molar ratio of (3-FL+2′-FL) to DFL of 3-10and a molar ratio of 3-FL to 2′-FL of 5-21 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 3:1 to 2:1 and a conversion rate of40-70%, an HMO mixture consisting essentially of 3-FL, 2′-FL, DFL andlactose, or an HMO mixture consisting essentially of 3-FL, 2′-FL andDFL, respectively, having a molar ratio of (3-FL+2′-FL) to DFL of 2-8and a molar ratio of 3-FL to 2′-FL of 2.5-7.7 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 2:1 to 1:1 and a conversion rate of35-55%, an HMO mixture consisting essentially of 3-FL, 2′-FL, DFL andlactose, or an HMO mixture consisting essentially of 3-FL, 2′-FL andDFL, respectively, having a molar ratio of (3-FL+2′-FL) to DFL of1.6-6.6 and a molar ratio of 3-FL to 2′-FL of 1-3.3 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 1:1 to 1:2 and a conversion rate of35-55%, an HMO mixture consisting essentially of 3-FL, 2′-FL, DFL andlactose, or an HMO mixture consisting essentially of 3-FL, 2′-FL andDFL, respectively, having a molar ratio of (3-FL+2′-FL) to DFL of1.6-6.6 and a molar ratio of 3-FL to 2′-FL of 0.3-1 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 1:2 to 1:3 and a conversion rate of40-70%, an HMO mixture consisting essentially of 3-FL, 2′-FL, DFL andlactose, or an HMO mixture consisting essentially of 3-FL , 2′-FL andDFL, respectively, having a molar ratio of (3-FL+2′-FL) to DFL of 2-8and a molar ratio of 3-FL to 2′-FL of 0.13-0.4 can be made.

When a process of the third or the fourth aspect is carried out with amolar ratio of 3-FL to 2′-FL of 1:3 to 1:5 and a conversion rate of50-80%, an HMO mixture consisting essentially of 3-FL, 2′-FL, DFL andlactose, or an HMO mixture consisting essentially of 3-FL, 2′-FL andDFL, respectively, having a molar ratio of (3-FL+2′-FL) to DFL of 3-10and a molar ratio of 3-FL to 2′-FL of 0.05-0.2 can be made.

The α1-3/4 Transfucosidase Suitable for Carrying Out the Third and theFourth Aspect of the Invention

In the following paragraphs, the expression “may carry” is equivalentwith the expression “optionally carries”, and the expression “can besubstituted” is equivalent with the expression “is optionallysubstituted”.

In accordance with this invention, the term “α1-3/4 transfucosidase”preferably means any wild type or engineered fucosidase that is able totransfer a fucose residue to the 3-position of the glucose in anacceptor of formula 2, to the 3-position of the N-acetyl-glucosamine ina, preferably terminal, N-acetyl-lactosaminyl group in an acceptor offormula 1, 1a or 1b, or to the 4-position of the N-acetyl-glucosamine ina, preferably terminal, lacto-N-biosyl group, in an acceptor of formula1, 1a or 1b, where the compounds of formulae 1 and 2 are as follows:

-   -   wherein R₁ is fucosyl or H,    -   R₂ is selected from N-acetyl-lactosaminyl and lacto-N-biosyl        groups, wherein the N-acetyl lactosaminyl group may carry a        glycosyl residue comprising one or more N-acetyl-lactosaminyl        and/or one or more lacto-N-biosyl groups; any        N-acetyl-lactosaminyl and lacto-N-biosyl group can be        substituted with one or more sialyl and/or fucosyl residue,    -   R₃ is H or N-acetyl-lactosaminyl group optionally substituted        with a glycosyl residue comprising one or more        N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups;        any N-acetyl-lactosaminyl and lacto-N-biosyl group can be        substituted with one or more sialyl and/or fucosyl residue, and    -   each R₄ independently is sialyl or H,    -   with the proviso that at least one of R₁ or R₄ is not H;        and the compounds of formulae 1a and 1b are as follows:

-   -   wherein R₁ is as defined above,    -   R_(2a) is an N-acetyl-lactosaminyl group optionally substituted        with a glycosyl residue comprising one N-acetyl-lactosaminyl        and/or one lacto-N-biosyl group; any N-acetyl-lactosaminyl and        lacto-N-biosyl group can be substituted with one or more sialyl        and/or fucosyl residue, but preferably void of a sialyl and/or        fucosyl residue,    -   R_(3a) is H or an N-acetyl-lactosaminyl group optionally        substituted with a lacto-N-biosyl group; any        N-acetyl-lactosaminyl and lacto-N-biosyl group can be        substituted with one or more sialyl and/or fucosyl residue, but        preferably void of a sialyl and/or fucosyl residue,    -   R_(2b) is a lacto-N-biosyl group optionally substituted with        sialyl and/or fucosyl residue, but preferably void of a sialyl        and/or fucosyl residue, and    -   R_(3b) is H or an N-acetyl-lactosaminyl group optionally        substituted with one or two N-acetyl-lactosaminyl and/or one        lacto-N-biosyl group; any N-acetyl-lactosaminyl and        lacto-N-biosyl group can be substituted with one or more sialyl        and/or fucosyl residue, but preferably without a sialyl and/or        fucosyl residue.        Preferably, the compounds of formulae 1a and 1b have one or more        of the following linkages and modifications:    -   the N-acetyl-lactosaminyl group in the glycosyl residue of        R_(2a) in formula 1a is attached to the another        N-acetyl-lactosaminyl group by a 1-3 interglycosidic linkage,    -   the lacto-N-biosyl group in the glycosyl residue of R_(2a) in        formula 1a is attached to the N-acetyl-lactosaminyl group by a        1-3 interglycosidic linkage, the lacto-N-biosyl group in the        glycosyl residue of R_(3a) in formula 1a is attached to the        N-acetyl-lactosaminyl group by a 1-3 interglycosidic linkage,    -   the N-acetyl-lactosaminyl group in the glycosyl residue of        R_(3b) in formula 1b is attached to another        N-acetyl-lactosaminyl group by a 1-3 or 1-6 interglycosidic        linkage, and    -   the lacto-N-biosyl group in the glycosyl residue of R_(3b) in        formula 1b is attached to the N-acetyl-lactosaminyl group by a        1-3 interglycosidic linkage.

The α1-3/4 transfucosidase is preferably selected from α-L-fucosidasesas classified according to EC 3.2.1.111, having transfucosidaseactivity, such as the α1-3/4 fucosidase from Bifidobacterium longumsubsp. infantis ATCC 15697 as set forth in U.S. Pat. No. 8,361,756 asprotein of SEQ ID No. 18 (SEQ ID No. 1 of the present application) andother fucosidases which have at least 60%, preferably at least 70%, morepreferably at least 80%, particularly at least 90%, identity with aminoacid positions 56 to 345 of the α1-3/4 fucosidase from Bifidobacteriumlongum subsp. infantis ATCC 15697. Examples of such other fucosidasesare listed below in Table 1.

TABLE 1 Description Accession No. α-L-fucosidase [Bifidobacterium longumsubsp. KEY30716.1 infantis EK3] α-L-fucosidase [Bifidobacterium longum]WP_013140205.1 putative α1-3/4 fucosidase [Bifidobacterium KFI63931.1kashiwanohense JCM 15439] putative α1-3/4 fucosidase [Bifidobacteriumscardovii] KFI94501.1 α-L-fucosidase [Gardnerella vaginalis]WP_004574432.1 α-L-fucosidase [Gardnerella vaginalis] WP_019261748.1hypothetical protein [Gardnerella vaginalis] WP_020759655.1α-L-fucosidase [Gardnerella vaginalis] WP_009993891.1 α-L-fucosidase[Gardnerella vaginalis] WP_004573610.1 α-L-fucosidase [Gardnerellavaginalis] WP_004120276.1 α-L-fucosidase [Gardnerella vaginalis]WP_004114072.1 α-L-fucosidase [Gardnerella vaginalis] WP_004137675.1α-L-fucosidase [Gardnerella vaginalis] WP_014554869.1 α-L-fucosidase[Bifidobacterium bifidum CAG: 234] WP_022173522.1 α-L-fucosidase[Actinomyces sp. ICM47] WP_009647833.1 hypothetical protein[Streptomyces ipomoeae] WP_009295550.1 α-L-fucosidase [Actinomyces sp.oral taxon 180] WP_009211856.1 hypothetical protein [Actinomyces sp.oral taxon 172] WP_021611755.1 α-L-fucosidase [Bifidobacterium sp. 7101]WP_029678277.1 hypothetical protein [Actinomyces sp. HPA0247]WP_016461038.1 α-L-fucosidase [Actinomyces sp. ICM54] EW096238.1α-L-fucosidase [Actinomyces odontolyticus] WP_003795385.1 α-L-fucosidase[Atopobium sp. ICM58] WP_009055210.1 α-L-fucosidase [Paenibacillus sp.J14] WP_028538247.1 α-L-fucosidase [Actinomyces odontolyticus]WP_003792781.1 α1-3/4 fucosidase [Propionibacterium acidipropionici]WP_015071771.1 α-L-fucosidase [Propionibacterium acidipropionici]WP_028700846.1 hypothetical protein [Paenibacillus barengoltzii]WP_016312877.1 α-L-fucosidase [Actinomyces sp. ICM39] WP_007588699.1α-L-fucosidase [Propionibacterium jensenii] WP_028703334.1α-L-fucosidase [Lactobacillus shenzhenensis] WP_022529554.1 hypotheticalprotein [Paenibacillus sp. HW567] WP_019912449.1 putative α-13/4-fucosidase [Clostridium hathewayi WP_022032399.1 CAG: 224]α-fucosidase [Clostridium hathewayi] WP_006775425.1 α-L-fucosidase[Janibacter sp. HTCC2649] WP_009776262.1 α-fucosidase [Clostridiumphytofermentans] WP_012201036.1 α-L-fucosidase [Enterococcus gallinarum]WP_029486307.1 uncharacterized protein [Blautia sp. CAG: 237]WP_022215646.1 MULTISPECIES: α-L-fucosidase [Enterococcus]WP_005470131.1 α-L-fucosidase [Enterococcus gallinarum EG2] EEV33648.1α-L-fucosidase [Ruminococcus sp. CAG: 60] CCY33010.1 α-L-fucosidase[Ruminococcus sp. CAG: 9] WP_022380664.1 α-fucosidase [Blautia wexlerae]WP_025580031.1 α-fucosidase [Ruminococcus sp. 5_1_39BFAA] WP_008706707.1α-fucosidase [Paenibacillus sp. HGF5] WP_009593620.1 α-L-fucosidase[Paenibacillus sp. FSL H8-457] ETT68114.1 hypothetical protein[Clostridium hathewayi] WP_002604401.1 hypothetical protein[Paenibacillus sp. PAMC 26794] WP_017691196.1 α-L-fucosidase[Paenibacillus sp. FSL R5-192] ETT29638.1 α-fucosidase [Paenibacillussp. Y412MC10] WP_015736742.1 α-L-fucosidase [Paenibacillus alvei]WP_021262981.1 α-fucosidase [Paenibacillus sp. UNC217MF] WP_028532504.1α-fucosidase [Paenibacillus alvei] WP_005546194.1 α-L-fucosidase[Paenibacillus alvei] WP_021254840.1 hypothetical protein [Paenibacillusterrigena] WP_018756045.1 α-fucosidase [Ruminococcus obeum]WP_005422251.1 α-L-fucosidase [Paenibacillus sp. FSL H7-689] ETT43086.1α-fucosidase [Paenibacillus lactis] WP_007127626.1 α-fucosidase[Bacillus sp. J13] WP_028406965.1 hypothetical protein [Paenibacillusdaejeonensis] WP_020617104.1 hypothetical protein [Clostridium sp. KLE1755] WP_021638714.1 α-fucosidase [Clostridium sp. ASBs410]WP_025233568.1 α-fucosidase [Paenibacillus vortex] WP_006211772.1α-L-fucosidase [Paenibacillus sp. FSL R5-808] ETT35249.1 α-fucosidase[Clostridium celerecrescens] KEZ90324.1 α-L-fucosidase [Firmicutesbacterium CAG: 94] WP_022336739.1 α-fucosidase [Clostridiales bacteriumVE202-27] WP_025488431.1 α-fucosidase [Paenibacillus pasadenensis]WP_028597616.1 MULTISPECIES: α-fucosidase [Paenibacillus] WP_024629466.1α-fucosidase [Paenibacillus sp. UNC451MF] WP_028551519.1 α-fucosidase[Paenibacillus sp. PAMC 26794] WP_026081066.1 α-fucosidase[Paenibacillus sp. JDR-2] WP_015843379.1 MULTISPECIES: α-fucosidase[Clostridiales] WP_009250084.1 α-fucosidase [Clostridiumsaccharolyticum] WP_013273060.1

It is important that the transfucosidase activity of the α1-3/4transfucosidase is greater than its hydrolytic activity. In the courseof the 3-FL+component B

component C+Lac or DFL+component E

component F+2′-FL reaction the hydrolysis of the component C orcomponent F can become significant at a certain time point, due to theincreasing concentration of the component C or component F, which arethen degraded into component B or component E, respectively, and fucose.In order to prepare the HMO mixtures of the invention, the reactionshould be stopped before there is significant product hydrolysis. Thistime point can be easily determined by well-known enzyme kineticmeasurements.

Especially preferred α1-3/4 transfucosidases for making the HMO mixturesof this invention are the α1-3/4 transfucosidases that lack hydrolyticactivity, or at least have significantly reduced hydrolytic activity.Such enzymes can be made by altering the amino acid sequence of a mainlywild type, α1-3/4 fucosidase at one or more amino acid positions, sothat the mutated amino acid sequence results in improved transfucosidaseactivity and/or reduced hydrolytic activity. In accordance with thisinvention, the α1-3/4 transfucosidase preferably:

-   -   i) has been mutated at least at one or more of the following        amino acid positions of SEQ ID No. 18 of U.S. Pat. No.        8,361,756: 134, 135, 168, 170, 174, 216, 221, 236, 237, 244,        245, 282 and 413, preferably at least at one or more of the        following amino acid positions: 134, 135, 174, 216, 221, 282 and        413; and thereby    -   ii) provides a conversion rate of at least 10% up to 70%,        preferably at least 20 %, more preferably at least 35%, even        more preferably 40-50%, for the reaction of        -   the 3-FL donor with the LNT acceptor, or        -   the 3-FL donor with the LNnT acceptor, or        -   the 3-FL donor with the LNFP-I acceptor, or        -   the 3-FL donor with the 2′-FL acceptor, or        -   the DFL donor with the LNT acceptor, or        -   the DFL donor with the LNnT acceptor, or        -   the DFL donor with the LNFP-I acceptor.

The Fifth Aspect of the Invention

The fifth aspect of this invention is an anti-infective composition fortreating bacterial infections. The composition comprises, preferablyconsists essentially of:

-   -   DFL,    -   a component E which is LNT, LNnT or LNFP-I,    -   a component F, which is:        -   LNFP-II when component E is LNT, or        -   LNFP-III when component E is LNnT, or        -   LNDFH-I when component E is LNFP-I, and    -   2′-FL,        or comprises, preferably consists essentially of:    -   3-FL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL, and    -   a fucosylated component C, which is:        -   LNFP-II when component B is LNT, or        -   LNFP-III when component B is LNnT, or        -   LNDFH-I when component B is LNFP-I, or        -   DFL when component B is 2′-FL;            or the composition consists essentially of    -   3-FL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL,    -   a fucosylated component C, which is        -   LNFP-II when component B is LNT, or        -   LNFP-III when component B is LNnT, or        -   LNDFH-I when component B is LNFP-I, or        -   DFL when component B is 2′-FL, and    -   lactose;        preferably the anti-infective composition consists essentially        of    -   3-FL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL, and    -   a component C, which is        -   LNFP-II when component B is LNT, or        -   LNFP-III when component B is LNnT, or        -   LNDFH-I when component B is LNFP-I, or        -   DFL when component B is 2′-FL,            or the anti-infective composition consists essentially of    -   DFL,    -   a component E which is LNT, LNnT or LNFP-I,    -   a component F, which is        -   LNFP-II when component E is LNT, or        -   LNFP-III when component E is LNnT, or        -   LNDFH-I when component E is LNFP-I, and    -   2′-FL.

The especially preferred anti-infective composition is selected from thegroup consisting of:

-   -   a mixture of HMOs consisting essentially of 3-FL, LNT and        LNFP-II or of 3-FL, LNT, LNFP-II and lactose, preferably in        which:        -   the molar ratio of (3-FL+LNT) relative to LNFP-II is            0.8-9.5, and        -   the molar ratio of lactose relative to LNFP-II is about 1,    -   provided that one of the molar ratios 3-FL to LNFP-II and LNT to        LNFP-II is not more than 2, particularly a mixture of HMOs        consisting essentially of 3-FL, LNT and LNFP-II; or    -   a mixture of HMOs consisting essentially of 3-FL, LNnT and        LNFP-III or of 3-FL, LNnT, LNFP-III and lactose, preferably in        which:        -   the molar ratio of (3-FL+LNnT) relative to LNFP-III is            0.8-9.5 and        -   the molar ratio of lactose relative to LNFP-III is about 1,    -   provided that one of the molar ratios 3-FL to LNFP-III and LNnT        to LNFP-III is not more than 2, particularly a mixture of HMOs        consisting essentially of 3-FL, LNnT and LNFP-III; or    -   a mixture of HMOs consisting essentially of 3-FL, LNFP-I and        LNDFH-I or of 3-FL, LNFP-I, LNDFH-I and lactose, preferably in        which:        -   the molar ratio of (3-FL+LNFP-I) relative to LNDFH-I is            0.8-9.5 and        -   the molar ratio of lactose relative to LNDFH-I is about 1,    -   particularly a mixture of HMOs consisting essentially of 3-FL,        LNFP-I and LNDFH-I; or    -   a mixture of HMOs consisting essentially of 3-FL, 2′-FL and DFL        or of 3-FL, 2′-FL, DFL and lactose, preferably in which:        -   the molar ratio of (3-FL+2′-FL) relative to DFL is 0.8-9.5            and        -   the molar ratio of lactose relative to DFL is about 1; or    -   a mixture of HMOs consisting essentially of 3-FL, 2′-FL and DFL,        preferably in which:        -   the molar ratio of DFL relative to (3-FL+2′-FL) is at least            1:10, preferably at least 1:7, more preferably at least 1:5,            especially 1:3; and/or        -   the molar ratio of 3-FL relative to 2′-FL is 0.05-21,            preferably 0.13-7.7, more preferably about 1; or    -   a mixture of HMOs comprising 2′-FL, DFL, LNT and LNFP-II,        preferably a mixture of HMOs consisting essentially of 2′-FL,        DFL, LNT and LNFP-II; or    -   a mixture of HMOs comprising 2′-FL, DFL, LNnT and LNFP-III -II,        preferably a mixture of HMOs consisting essentially of 2′-FL,        DFL, LNnT and LNFP-III; or    -   a mixture of HMOs comprising 2′-FL, DFL, LNFP-I and LNDFH-I,        preferably a mixture of HMOs consisting essentially of 2′-FL,        DFL, LNFP-I and LNDFH-I.

The anti-infective composition of this invention can be a pharmaceuticalcomposition. The pharmaceutical composition can contain apharmaceutically acceptable carrier, e.g. phosphate buffered salinesolution, mixtures of ethanol in water, water and emulsions such as anoil/water or water/oil emulsion, as well as various wetting agents orexcipients. The pharmaceutical composition can also contain othermaterials that do not produce an adverse, allergic or otherwise unwantedreaction when administered to a patient. The carriers and othermaterials can include solvents, dispersants, coatings, absorptionpromoting agents, controlled release agents, and one or more inertexcipients, such as starches, polyols, granulating agents,microcrystalline cellulose, diluents, lubricants, binders, anddisintegrating agents. If desired, tablet dosages of the anti-infectivecompositions can be coated by standard aqueous or non-aqueoustechniques.

The anti-infective compositions of this invention can be administeredorally, e.g. as a tablet, capsule, or pellet containing a predeterminedamount of the first mixture, or as a powder or granules containing apredetermined concentration of the first mixture or a gel, paste,solution, suspension, emulsion, syrup, bolus, electuary, or slurry, inan aqueous or non-aqueous liquid, containing a predeterminedconcentration of the first mixture. Orally administered compositions caninclude binders, lubricants, inert diluents, flavouring agents, andhumectants. Orally administered compositions such as tablets canoptionally be coated and can be formulated so as to provide sustained,delayed or controlled release of the first mixture therein.

The anti-infective compositions of this invention can also beadministered by rectal suppository, aerosol tube, naso-gastric tube ordirect infusion into the Gl tract or stomach.

Anti-infective pharmaceutical compositions of this invention can alsoinclude therapeutic agents such as antiviral agents, antibiotics,probiotics, analgesics, and anti-inflammatory agents. The proper dosageof these compositions for a patient can be determined in a conventionalmanner, based upon factors such as the patient's immune status, bodyweight and age. In some cases, the dosage will be at a concentrationsimilar to that found for the HMOs of the composition in human breastmilk. The required amount would generally be in the range from about 200mg to about 20 g per day, in certain embodiments from about 300 mg toabout 15 g per day, from about 400 mg to about 10 g per day, in certainembodiments from about 500 mg to about 10 g per day, in certainembodiments from about 1 g to about 10 g per day. Appropriate doseregimes can be determined by methods known to those skilled in the art.

The anti-infective compositions of this invention can also be added tonutritional compositions. For example, they can be added to an infantformula, a nutritional composition, a rehydration solution, or a dietarymaintenance or supplement for elderly individuals or immunocompromisedindividuals. Macronutrients such as edible fats, carbohydrates andproteins can also be included in such anti-infective compositions.Edible fats include, for example, coconut oil, soy oil andmonoglycerides and diglycerides. Carbohydrates include, for example,glucose, edible lactose and hydrolysed cornstarch. Proteins include, forexample, soy protein, whey, and skim milk. Vitamins and minerals (e. g.calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese,iron, copper, zinc, selenium, iodine, and Vitamins A, E, D, C, and Bcomplex) can also be included in such anti-infective compositions.

The Sixth Aspect of the Invention

The sixth aspect of the invention is a method of modulating themicrobiome of a human to increase Bifidobacterium abundance andBarnesiella abundance. Preferably, the method reduces Firmicutesabundance, especially Clostridia. The method comprises administering tothe human:

-   i) a composition comprising, preferably consisting essentially of,    -   3-FL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL, and    -   a component C, which is        -   LNFP-II when component B is LNT, or        -   LNFP-III when component B is LNnT, or        -   LNDFH-I when component B is LNFP-I, or        -   DFL when component B is 2′-FL;            or-   ii) a composition consisting essentially of    -   3-FL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL, and    -   a component C, which is        -   LNFP-II when component B is LNT, or        -   LNFP-III when component B is LNnT, or        -   LNDFH-I when component B is LNFP-I, or        -   DFL when component B is 2′-FL; and    -   lactose;        or-   iii) a composition comprising, preferably consisting essentially of,    -   DFL,    -   a component E which is LNT, LNnT or LNFP-I,    -   a component F, which is        -   LNFP-II when component E is LNT, or        -   LNFP-III when component E is LNnT, or        -   LNDFH-I when component E is LNFP-I, and    -   2′-FL,        preferably a composition consisting essentially of    -   3-FL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL, and    -   a component C, which is        -   LNFP-II when component B is LNT, or        -   LNFP-III when component B is LNnT, or        -   LNDFH-I when component B is LNFP-I, or        -   DFL when component B is 2′-FL,            or a composition consisting essentially of    -   DFL,    -   a component E which is LNT, LNnT or LNFP-I,    -   a component F, which is        -   LNFP-II when component E is LNT, or        -   LNFP-III when component E is LNnT, or        -   LNDFH-I when component E is LNFP-I, and    -   2′-FL.

Especially preferred methods for modulating the microbiome of a humaninvolve administering to the human:

-   -   3-FL, LNT and LNFP-II, preferably a mixture of HMOs consisting        essentially of    -   3-FL, LNT and LNFP-II or of 3-FL, LNT, LNFP-II and lactose, more        preferably in which:        -   the molar ratio of (3-FL+LNT) relative to LNFP-II is            0.8-9.5, and        -   the molar ratio of lactose relative to LNFP-II is about 1,    -    provided that one of the molar ratios 3-FL to LNFP-II and LNT        to LNFP-II is not more than 2, particularly a mixture of HMOs        consisting essentially of 3-FL, LNT and LNFP-II; or    -   3-FL, LNnT and LNFP-III, preferably a mixture of HMOs consisting        essentially of 3-FL, LNnT and LNFP-III or of 3-FL, LNnT,        LNFP-III and lactose, more preferably in which:        -   the molar ratio of (3-FL+LNnT) relative to LNFP-III is            0.8-9.5 and        -   the molar ratio of lactose relative to LNFP-III is about 1,    -    provided that one of the molar ratios 3-FL to LNFP-III and LNnT        to LNFP-III is not more than 2, particularly a mixture of HMOs        consisting essentially of 3-FL, LNnT and LNFP-III; or    -   3-FL, LNFP-I and LNDFH-I, preferably a mixture of HMOs        consisting essentially of 3-FL, LNFP-I and LNDFH-I or of 3-FL,        LNFP-I, LNDFH-I and lactose, more preferably in which:        -   the molar ratio of (3-FL+LNFP-I) relative to LNDFH-I is            0.8-9.5 and        -   the molar ratio of lactose relative to LNDFH-I is about 1,    -    particularly a mixture of HMOs consisting essentially of 3-FL,        LNFP-I and LNDFH-I; or    -   3-FL, 2′-FL and DFL, preferably a mixture of HMOs consisting        essentially of 3-FL, 2′-FL and DFL or of 3-FL, 2′-FL, DFL and        lactose, more preferably in which:        -   the molar ratio of (3-FL+2′-FL) relative to DFL is 0.8-9.5            and        -   the molar ratio of lactose relative to DFL is about 1,    -    particularly a mixture of HMOs consisting essentially of 3-FL,        2′-FL and DFL; or    -   2′-FL, DFL, LNT and LNFP-II, preferably a mixture of HMOs        comprising 2′-FL, DFL, LNT and LNFP-II, more preferably a        mixture of HMOs consisting essentially of2′-FL, DFL, LNT and        LNFP-II; or    -   2′-FL, DFL, LNnT and LNFP-III, preferably a mixture of HMOs        comprising 2′-FL, DFL, LNnT and LNFP-III -II, more preferably a        mixture of HMOs consisting essentially of2′-FL, DFL, LNnT and        LNFP-III; or    -   2′-FL, DFL, LNFP-I and LNDFH-I, preferably a mixture of HMOs        comprising 2′-FL, DFL, LNFP-I and LNDFH-I, more preferably a        mixture of HMOs consisting essentially of 2′-FL, DFL, LNFP-I and        LNDFH-I.

The Seventh Aspect of the Invention

The seven aspect of the invention is a method of preventing or treatingbacterial infections in a human, especially antibiotic resistantbacterial infections. The method comprises administering to the human,

-   i) a composition comprising, preferably consisting essentially of,    -   3-FL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL, and    -   a fucosylated component C, which is        -   LNFP-II when component B is LNT, or        -   LNFP-III when component B is LNnT, or        -   LNDFH-I when component B is LNFP-I, or        -   DFL when component B is 2′-FL;            or-   ii) a composition consisting essentially of    -   3-FL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL, and    -   a component C, which is        -   LNFP-II when component B is LNT, or        -   LNFP-III when component B is LNnT, or        -   LNDFH-I when component B is LNFP-I, or        -   DFL when component B is 2′-FL; and    -   lactose;        or-   iii) a composition comprising, preferably consisting essentially of,    -   DFL,    -   a component E which is LNT, LNnT or LNFP-I,    -   a component F, which is        -   LNFP-II when component E is LNT, or        -   LNFP-III when component E is LNnT, or        -   LNDFH-I when component E is LNFP-I, and    -   2′-FL,        preferably a composition consisting essentially of    -   3-FL,    -   a component B which is LNT, LNnT, LNFP-I or 2′-FL, and    -   a component C, which is        -   LNFP-II when component B is LNT, or        -   LNFP-III when component B is LNnT, or        -   LNDFH-I when component B is LNFP-I, or        -   DFL when component B is 2′-FL,            or a composition consisting essentially of    -   DFL,    -   a component E which is LNT, LNnT or LNFP-I,    -   a component F, which is        -   LNFP-II when component E is LNT, or        -   LNFP-III when component E is LNnT, or        -   LNDFH-I when component E is LNFP-I, and    -   2′-FL.

Especially preferred methods for preventing or treating bacterialinfections in human comprises administering to the human:

-   -   3-FL, LNT and LNFP-II, preferably a mixture of HMOs consisting        essentially of 3-FL, LNT and LNFP-II or of 3-FL, LNT, LNFP-II        and lactose, more preferably in which:        -   the molar ratio of (3-FL+LNT) relative to LNFP-II is            0.8-9.5, and        -   the molar ratio of lactose relative to LNFP-II is about 1,    -    provided that one of the molar ratios 3-FL to LNFP-II and LNT        to LNFP-II is not more than 2, particularly a mixture of HMOs        consisting essentially of 3-FL, LNT and LNFP-II; or    -   3-FL, LNnT and LNFP-III, preferably a mixture of HMOs consisting        essentially of 3-FL, LNnT and LNFP-III or of 3-FL, LNnT,        LNFP-III and lactose, more preferably in which:        -   the molar ratio of (3-FL+LNnT) relative to LNFP-III is            0.8-9.5 and        -   the molar ratio of lactose relative to LNFP-III is about 1,    -    provided that one of the molar ratios 3-FL to LNFP-III and LNnT        to LNFP-III is not more than 2, particularly a mixture of HMOs        consisting essentially of 3-FL, LNnT and LNFP-III; or    -   3-FL, LNFP-I and LNDFH-I, preferably a mixture of HMOs        consisting essentially of 3-FL, LNFP-I and LNDFH-I or of 3-FL,        LNFP-I, LNDFH-I and lactose, more preferably in which:        -   the molar ratio of (3-FL+LNFP-I) relative to LNDFH-I is            0.8-9.5 and        -   the molar ratio of lactose relative to LNDFH-I is about 1,    -    particularly a mixture of HMOs consisting essentially of 3-FL,        LNFP-I and LNDFH-I; or    -   3-FL, 2′-FL and DFL, preferably a mixture of HMOs consisting        essentially of 3-FL, 2′-FL and DFL or of 3-FL, 2′-FL, DFL and        lactose, more preferably in which:        -   the molar ratio of (3-FL+2′-FL) relative to DFL is 0.8-9.5            and        -   the molar ratio of lactose relative to DFL is about 1,    -    particularly a mixture of HMOs consisting essentially of 3-FL,        2′-FL and DFL; or    -   2′-FL, DFL, LNT and LNFP-II, preferably a mixture of HMOs        comprising 2′-FL, DFL, LNT and LNFP-II, more preferably a        mixture of HMOs consisting essentially of2′-FL, DFL, LNT and        LNFP-II; or    -   2′-FL, DFL, LNnT and LNFP-III, preferably a mixture of HMOs        comprising 2′-FL, DFL, LNnT and LNFP-III -II, more preferably a        mixture of HMOs consisting essentially of2′-FL, DFL, LNnT and        LNFP-III; or    -   2′-FL, DFL, LNFP-I and LNDFH-I, preferably a mixture of HMOs        comprising 2′-FL, DFL, LNFP-I and LNDFH-I, more preferably a        mixture of HMOs consisting essentially of2′-FL, DFL, LNFP-I and        LNDFH-I.

Whilst the invention has been described with reference to preferredembodiments, it will be appreciated that various modifications arepossible within the scope of the invention.

In this specification, unless expressly otherwise indicated, the word‘or’ is used in the sense of an operator that returns a true value wheneither or both of the stated conditions is met, as opposed to theoperator ‘exclusive or’ which requires that only one of the conditionsis met. The word ‘comprising’ is used in the sense of ‘including’ ratherthan in to mean ‘consisting of’. All prior teachings acknowledged aboveare hereby incorporated by reference. No acknowledgement of any priorpublished document herein should be taken to be an admission orrepresentation that the teaching thereof was common general knowledge inAustralia or elsewhere at the date hereof.

EXAMPLES

In the examples below, mutants of Bifidobacterium longum subsp. infantisATCC 15697 were used for making mixtures of this invention; thepositions of mutations are with reference to SEQ ID No. 1 of thisapplication, which is identical with SEQ ID No. 18 of U.S. Pat. No.8,361,756.

Example 1

3-FL+LNT

LNFP-II+Lac

The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200μl), [3-FL]=200 mM, [LNT]=200 mM, enzyme extract=0.5 mg/ml. HPLCconditions: TSK Gel amide 80 (Tosoh, 3 μm, 150×4.6mm) was used with aflow of 1 ml/min using 56% acetonitrile and 44 % water. The elution ofsubstrates and products was detected by CAD and/or UV detection at 195nm.

The table below shows the composition of mixtures obtained. Lactose isequimolar to LNFP-II.

molar ratio mutant conversion, time 3-FL LNT LNFP-II N216D 43.9%, 15 min1.28 1.28 1 V221A 35.6%, 15 min 1.81 1.81 1 V282K 39.7%, 4 h 1.52 1.52 1P134A 36.4%, 4 h 1.75 1.75 1 W135F 44.5%, 15 min 1.25 1.25 1 W135A39.5%, 2 h 1.53 1.53 1 W135A 46.1%, 6.5 h 1.17 1.17 1 W135E 39.4%, 15min 1.54 1.54 1 W135E 43.2%, 30 min 1.31 1.31 1 W135E 45.2%, 1 h 1.211.21 1 A174S 45.1%, 15 min 1.22 1.22 1 Q244K 37.6%, 15 min 1.66 1.66 1Q244K 45.1%, 1 h 1.22 1.22 1 Q244K 50.5%, 6.5 h 0.98 0.98 1S168E-A174H-E413R 41.1%, 2 h 1.43 1.43 1 S168E-A174H-E413R 48.7%, 23 h1.05 1.05 1 S168E-A174H-V282E 37.8%, 1 h 1.65 1.65 1 S168E-A174H-V282E48.5%, 6 h 1.06 1.06 1 S168E-A174H-V221A- 48.5%, 6 h 1.06 1.06 1 V282HW135E-A174F-V221A 47.4%, 6 h 1.11 1.11 1

Example 2

The same test was carried out, using the W135F-A174N-N274A-E413 mutant,as in Example 1 with the difference that the 3-FL: LNT ratio varied from3:1 to 1:3, and the enzyme extract was 0.05 mg/ml). The table belowshows the composition of mixtures obtained. Lactose is equimolar toLNFP-II. No fucose was detected.

molar ratio 3-FL/LNT ratio conversion, time 3-FL LNT LNFP-II 3:1 42%, 15min 6.14 1.38 1 3:1 48%, 30 min 5.25 1.08 1 3:1 57%, 1 h 4.26 0.75 1 3:165%, 2 h 3.62 0.54 1 3:1 66%, 6.5 h 3.55 0.52 1 2:1 34%, 30 min 4.881.94 1 2:1 43%, 1 h 3.65 1.33 1 2:1 57%, 4 h 2.51 0.75 1 1:1 34%, 2 h1.94 1.94 1 1:1 43%, 6.5 h 1.33 1.33 1 1:2 40%, 1 h 1.5 4 1 1:2 49%, 2 h1.04 3.08 1 1:2 55%, 3 h 0.82 2.64 1 1:2 60%, 9 h 0.67 2.33 1 1:3 39%,15 min 1.56 6.69 1 1:3 53%, 1 h 0.89 4.66 1 1:3 62%, 2 h 0.61 3.84 1 1:369%, 6.5 h 0.45 3.35 1

Example 3

3-FL+LNnT

LNFP-III+Lac

The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200μl), [3-FL]=200 mM, [LNnT]=200 mM,. HPLC conditions: TSK Gel amide 80(Tosoh, 3 μm, 150×4.6 mm) was used with a flow of 1 ml/min using 56%acetonitrile and 44% water. The elution of substrates and products wasdetected by CAD and/or UV detection at 195 nm.

The table below shows the composition of mixtures obtained for an enzymeextract of 2 mg/ml. Lactose is equimolar to LNFP-III.

molar ratio mutant conversion, time 3-FL LNnT LNFP-III N216D 29.73%, 15min 2.36 2.36 1 V221A 24.19%, 15 min 3.13 3.13 1 V282K  38.1%, 1 h 1.621.62 1 P134A 40.46%, 15 min 1.47 1.47 1 W135F 44.57%, 1 h 1.24 1.24 1W135A 45.58%, 15 min 1.19 1.19 1 W135E 44.19%, 1 h 1.26 1.26 1 W170F40.06%, 15 min 1.50 1.50 1 A174S  38.4%, 15 min 1.60 1.60 1 A174H 49.3%, 2 h 1.03 1.03 1

The table below shows the composition of mixtures obtained for an enzymeextract of 0.5 mg/ml. Lactose is equimolar to LNFP-III.

molar ratio mutant conversion, time 3-FL LNnT LNFP-III N216D 29.00%, 15min 2.45 2.45 1 V221A 26.00%, 15 min 2.85 2.85 1 V282K 27.00%, 4 h 2.702.70 1 P134A 32.00%, 1 h 2.13 2.13 1 W135F 34.20%, 4 h 1.92 1.92 1 W135A36.00%, 2 h 1.78 1.78 1 W135E 36.30%, 4 h 1.75 1.75 1 W170F 32.00%, 2 h2.13 2.13 1 A174S 27.30%, 2 h 2.66 2.66 1 A174H 44.00%, 6.5 h 1.27 1.271 Q244K 22.50%, 15 min 3.44 3.44 1

Example 4

The same test was carried out as in Example 3 using multi-point mutants.The table below shows the composition of mixtures obtained for an enzymeextract of 0.5 mg/ml. Lactose is equimolarto LNFP-III.

molar ratio mutant conversion, time 3-FL LNnT LNFP-III S168E-A174H-E413R51.15%, 23 h 0.96 0.96 1 S168E-A174F   49.16, 23 h 1.03 1.03 1S168E-A174H-V282E 51.55%, 6 h 0.94 0.94 1 S168E-A174H-V221A  51.0%, 23 h0.96 0.96 1 S168E-A174H-V221A- 51.08%, 6 h 0.96 0.96 1 V282HW135E-A174F-V221A 52.77%, 23 h 0.90 0.90 1 S168E-A174F-V221A- 52.12%, 23h 0.92 0.92 1 V282R S168E-A174H-N216D 51.65%, 23 h 0.94 0.94 1W135E-A174F-N216D- 50.01%, 6 h 1 1 1 V221A S168E-A174H 51.08%, 23 h 0.960.96 1

Example 5

3-FL+LNFP-I

LNDFH-I+Lac

The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200μl), [3-FL]=200 mM, [LNFP-I]=200 mM, enzyme extract=0.5 mg/ml. HPLCconditions: TSK Gel amide 80 (Tosoh, 3 μm, 150×4.6 mm) was used with aflow of 1 ml/min using 56% acetonitrile and 44% water. The elution ofsubstrates and products was detected by CAD and/or UV detection at 195nm.

The following table below shows the composition of mixtures obtained.Lactose is equimolar to LNDFH-I.

molar ratio mutant conversion, time 3-FL LNFP-I LNDFH-I N216D  43.3%, 15min 1.31 1.31 1 V221A  35.5%, 15 min 1.82 1.82 1 V282K  36.8%, 25 h 1.721.72 1 P134A  45.3%, 2 h 1.21 1.21 1 W135F  51.9%, 25 h 0.93 0.93 1W135A  44.3%, 6.5 h 1.26 1.26 1 W135E  50.7%, 25 h 0.97 0.97 1 W170F 44.5%, 1 h 1.25 1.25 1 A174S  31.7%, 25 h 2.15 2.15 1 Q244K  42.8%, 15min 1.34 1.34 1 S168E-A174F 25.72%, 23 h 2.89 2.89 1 S168E-A174H-V221A24.75%, 23 h 3.04 3.04 1 W135E-A174F-V221A 41.21%, 23 h 1.43 1.43 1W135E-A174F-N2126D- 38.46%, 23 h 1.60 1.60 1 V221A

Another test was run in sodium phosphate buffer (50 mM, pH=6.5, 30° C.,140 μl), [3-FL]=50 mM, [LNFP-I]=50 mM, enzyme extract=10 μl.

The following table below shows the composition of mixtures obtained.Lactose is equimolar to LNDFH-I.

molar ratio mutant conversion, time 3-FL LNFP-I LNDFH-I P134A 33%, 15min 2.03 2.03 1 P134V 20%, 15 min 4.0 4.0 1 W135F 37%, 4.5 h 1.70 1.70 1W135A 25%, 4.5 h 3.0 3.0 1 W135E 37%, 4.5 h 1.70 1.70 1 W170F 33%, 15min 2.03 2.03 1 A236D 38%, 15 min 1.63 1.63 1 A236H 39%, 15 min 1.561.56 1 E237N 38%, 4.5 h 1.63 1.63 1 Q244L 42%, 15 min 1.38 1.38 1 Q244H42%, 15 min 1.38 1.38 1 Q244K 41%, 15 min 1.44 1.44 1 Q244R 31%, 15 min2.23 2.23 1 Q245E 36%, 15 min 1.78 1.78 1 W135E-A174F-A236E 50%, 4.5 h1.00 1.00 1 W135E-A174F-L238A 51%, 4.5 h 0.96 0.96 1 W135E-A174F-T239H53%, 4.5 h 0.89 0.89 1 W135E-A174F-E241H 52%, 4.5 h 0.92 0.92 1

Example 6

The same test was carried out as in Example 5 in sodium phosphate buffer(50 mM, pH=6.5, 37° C. 150 μl), [3-FL]=100 mM, [LNFP-I]=50 mM, enzymeextract=10 μl crude extract. HPLC was as in Example 5.

The following table below shows the composition of mixtures obtainedafter 220 minutes. Lactose is equimolar to LNDFH-I.

molar ratio mutant conversion 3-FL LNFP-I LNDFH-I W135E-A174F-E413R 40%, 4.0 1.5 1 W135Y-A174V-E413R 45% 3.44 1.22 1 W135F-A174F-E413R 41%3.88 1.44 1 W135Y-A174F-E413R 52% 2.85 0.92 1 W135Y-A174G-E413R 43% 3.651.33 1 W135F-A174N-E413R 58% 2.45 0.72 1 W135Y-A174N-E413R 57% 2.51 0.751 W135Q-A174N-E413R 56% 2.57 0.79 1 W135Y-A174S-E413R 47% 3.26 1.13 1W135F-A174S-E413R 52% 2.85 0.92 1

Example 7

3-FL+2′-FL

DFL+Lac

The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200μl), [3-FL]=200 mM, [2′-FL]=200 mM, enzyme extract=0.5 mg/ml. HPLCconditions: TSK Gel amide 80 (Tosoh, 3 μm, 150×4.6mm) was used with aflow of 1 ml/min using 56% acetonitrile and 44% water. The elution ofsubstrates and products was detected by CAD and/or UV detection at 195nm.

The table below shows the composition of mixtures obtained. Lactose isequimolar to DFL.

molar ratio mutant conversion, time 3-FL 2′-FL DFL N216D 46%, 30 min1.17 1.17 1 V282K 51%, 25 h 0.96 0.96 1 P134A 54%, 2 h 0.85 0.85 1 W135F59%, 25 h 0.69 0.69 1 W135A 52%, 6 h 0.92 0.92 1 W135E 59%, 25 h 0.690.69 1 A174S 41%, 4 h 1.44 1.44 1 A174H 30%, 25 H 2.33 2.33 1 Q244K 33%,30 min 2.03 2.03 1

Example 8

DFL+LNT

LNFP-II+2′-Lac

The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200μl), using the W135F-A174N-N274A-E413R mutant, [DFL]=200 mM, [LNT]=200mM, enzyme extract=0.1 or 0.25 mg/ml. HPLC conditions: TSK Gel amide 80(Tosoh, 3 μm, 150×4.6mm) was used with a flow of 1.1 ml/min using 66%acetonitrile and 34% water. The elution of substrates and products wasdetected by CAD and/or UV detection at 195 nm.

The tables below show the composition of mixtures obtained.

enzyme extract: 0.1 mg/ml

LNFP-II DFL LNT LNFP-II 2′-FL time (h) conversion (mol %) (mol %) (mol%) (mol %) 1 10%   45%   45%   5%   5% 2 15% 42.5% 42.5%  7.5%  7.5% 524%   38%   38%   12%   12% 8 27% 36.5% 36.5% 13.5% 13.5%

enzyme extract: 0.25 mg/ml

LNFP-II DFL LNT LNFP-II 2′-FL time (h) conversion (mol %) (mol %) (mol%) (mol %) 0.5 10% 45% 45% 5% 5% 1 16% 42% 42% 8% 8% 2 20% 40% 40% 10%10% 5 28% 36% 36% 14% 14% 8 28% 36% 36% 14% 14%

Example 9

DFL+LNT

LNFP-III+2′-FL

The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200μl), using the W135F-A174N-N274A-E413R mutant, [DFL]=200 mM, [LNnT]=200mM, enzyme extract=0.1 or 0.25 mg/ml. HPLC conditions: TSK Gel amide 80(Tosoh, 3 μm, 150×4.6mm) was used with a flow of 1.1 ml/min using 66%acetonitrile and 34% water. The elution of substrates and products wasdetected by CAD and/or UV detection at 195 nm.

The tables below show the composition of mixtures obtained.

enzyme extract: 0.1 mg/ml

LNFP-III DFL LNnT LNFP-III 2′-FL time (h) conversion (mol %) (mol %)(mol %) (mol %) 1 7% 46.5% 46.5% 3.5% 3.5% 2 11% 44.5% 44.5% 5.5% 5.5% 519% 40.5% 40.5% 9.5% 9.5% 8 21% 39.5% 39.5% 10.5% 10.5%

enzyme extract: 0.25 mg/ml

LNFP-III DFL LNnT LNFP-III 2′-FL time (h) conversion (mol %) (mol %)(mol %) (mol %) 0.5 8% 46% 46% 4% 4% 1 12% 44% 44% 6% 6% 2 17% 41.5%  41.5%   8.5%   8.5%   5 24% 38% 38% 12%  12%  8 25% 37.5%   37.5%  12.5%   12.5%  

Example 10

DFL+LNFP-I

LNDFH-I+2′-FL

The test was run in sodium phosphate buffer (50 mM, pH=6.5, 37° C., 200μl), using the W135F-A174N-N274A-E413R mutant, [DFL]=200 mM,[LNFP-I]=200 mM, enzyme extract=0.1 or 0.25 mg/ml. HPLC conditions: TSKGel amide 80 (Tosoh, 3 μm, 150×4.6mm) was used with a flow of 1.1 ml/minusing 66% acetonitrile and 34% water. The elution of substrates andproducts was detected by CAD and/or UV detection at 195 nm.

The tables below show the composition of mixtures obtained.

enzyme extract: 0.1 mg/ml

LNDFH-I DFL LNFP-I LNDFH-I 2′-FL time (h) conversion (mol %) (mol %)(mol %) (mol %) 1 11% 44.5% 44.5% 5.5% 5.5% 2 16%   42%   42%   8%   8%5 23% 38.5% 38.5% 11.5%  11.5%  8 32%   34%   34%  16%  16%

enzyme extract: 0.25 mg/ml

time LNDFH-I DFL LNFP-I LNDFH-I 2′-FL (h) conversion (mol %) (mol %)(mol %) (mol %) 0.5 11% 44.5%   44.5%   5.5%  5.5%  1 20% 40% 40% 10%10% 2 28% 36% 36% 14% 14% 5 36% 32% 32% 18% 18% 8 38% 31% 31% 19% 19%

Example 11

A total of 80 male and female patients are recruited to participate inthe study. After a screening visit and run-in period of 1-2 weeks, thepatients are selected and randomized into eight groups, each of 10patients. Seven groups are each administered a treatment productcontaining 5 grams of one of the following compositions:

i)LNT, 3-FL and LNFP-II,

ii) LNnT, 3-FL and LNFP-III,

iii) LNFP-I, 3-FL and LNDFH-I,

iv) 3-FL, 2′-FL and DFL,

v) 2′-FL, DFL, LNT and LNFP-II,

vi) 2′-FL, DFL, LNnT and LNFP-III, or

vii) 2′-FL, DFL, LNFP-I and LNDFH-I,

and one group the placebo product (2 grams of glucose) for 8 weeks. Thetreatment products and the placebo are in powder form in a unit dosagecontainer.

The patients are eligible to participate if they are at least 18 yearsof age. All recruited patients are able and willing to understand andcomply with the study procedures. Patients are excluded if: they haveparticipated in a clinical study one month prior to screening visit;they have abnormal results in the screening tests which are clinicallyrelevant for study participation; they are suffering for a severedisease such as malignancy, diabetes, severe coronary disease, kidneydisease, neurological disease, or severe psychiatric disease or anycondition which can confound the results of the study; used highly dosedprobiotic supplements (yoghurt allowed) for 3 months prior to the study;consumed antibiotic drugs 3 months prior to the study; consumed on aregular basis any medication that might interfere with symptomevaluation 2 weeks prior to the study; and pregnant or lactating.

At the screening visit, medical history and concomitant medication isregistered and a blood sample for safety analyses is collected. A faecalsample kit is distributed. Patients are instructed to keep their samplesin the freezer until the next visit.

At the second visit, eligibility criteria are checked and eligiblesubjects are randomised to the three arms in the trial. The faecalsamples are collected and equipment for new samples are distributed.Patients are familiarised with an interactive internet enabled systemwhich records data daily and are provided with either treatment orcontrol products. Subjects are reminded not to change their usual dietduring the study. Blood samples are collected for biomarker studies. Thefaecal samples are stored at −80° C. until analysis. Faecal samples aresubjected to 16 S RNA sequencing analysis.

The study runs for 8 weeks with the patients consuming either a placeboor a treatment product daily. Patients are instructed to consume theproducts in the morning with breakfast. Compliance is monitored throughthe interactive internet enabled system. The patients also use thesystem to record:

-   -   Bristol Stool Form Scale (BSF) information,    -   symptom information such as abdominal pain, abdominal        discomfort, abdominal cramping, abdominal bloating, and        abdominal fullness,    -   additional Gastrointestinal Symptom Rating Scale (GSRS)        information.

This questionnaire includes 15 items covering five dimensions (abdominalpain, indigestion, reflux, diarrhoea, constipation) and uses aseven-graded Likert scale.

At the end of the study, each patient has an exit visit with the medicalteam. Faecal samples and blood samples are collected and analysed asbefore.

The faecal analysis indicates that the treatment patients have increasedabundance of Bifidobacterium and Barnesiella, and reduced abundance ofFirmicutes; especially Clostridia.

Example 12

Eighty 7-week-old C57BL/6J female mice are individually housed to avoidcontamination between mice and provided with irradiated food and water.The mice are separated into 8 groups of 10 mice, 7 treatment groups anda placebo group.

The mice are treated with ampicillin (0.5 g/liter) in their drinkingwater, which is changed every 3 days. After 1 week, the ampicillinaddition to the drinking water is terminated. Thereafter one of thefollowing compositions:

i) LNT, 3-FL and LNFP-II

ii) LNnT, 3-FL and LNFP-III,

iii) LNFP-I, 3-FL and LNDFH-I,

iv) 3-FL, 2′-FL and DFL,

v) 2′-FL, DFL, LNT and LNFP-II,

vi) 2′-FL, DFL, LNnT and LNFP-III, or

vii) 2′-FL, DFL, LNFP-I and LNDFH-I,

is added to the drinking water of each of the seven treatment groups ata total concentration of 40 mg/ml. The control group receives plainwater. Fresh water is administered daily and all mice have free accessto the drinking water. The mice are fed a rodent chow and are givenfresh chow daily.

Two days after termination of the ampicillin treatment, mice of eachgroup is infected by means of oral gavage with a vancomycin-resistantEnterococcus faecium strain (VRE). VRE levels are determined atdifferent time points by plating serial dilutions of faecal pellets onEnterococcosel agar plates with vancomycin. VRE colonies are identifiedby appearance and confirmed by Gram staining. PCR of the vanA gene,which confers resistance to vancomycin, is used to confirm the presenceof VRE in infected mice.

The mice are monitored for 2 weeks and are then euthanized. Fresh stoolpellets are obtained before the mice are euthanized. The samples areimmediately frozen and stored at −80° C. DNA is extracted using a96-well PowerSoil DNA Isolation Kit (MO-BIO). A minimum of onesample-well per plate is kept empty to serve as a negative controlduring PCR. PCR is done with the forward primer S-D-Bact-0341-b-S-17 andreverse primer S-D-Bact-0785-a-A-21 (Klindworth et al. Nucleic AcidsRes. 41, e1 (2013)) with illumina adapters attached. These are universalbacterial 16S rDNA primers, which target the V3-V4 region. The followingPCR program is used: 98° C. for 30 sec, 25× (98° C. for 10 s, 55° C. for20 s, 72° C. for 20 s), 72° C. for 5 min. Amplification is verified byrunning the products on a 1% agarose gel. Barcodes are added in a nestedPCR using the Nextera Index Kit V2 (Illumina) with the following PCRprogram: 98° C. for 30 sec, 8× (98° C. for 10 s, 55° C. for 20 s, 72° C.for 20 s), 72° C. for 5 min. Attachment of primers is verified byrunning the products on a 1% agarose gel.

Products from the nested PCR are normalized using the SequalPrepNormalization Plate Kit and pooled. Pooled libraries are concentrated byevaporation and the DNA concentration of pooled libraries wisas measuredon a Qubit fluorometer using the Qubit High Sensitivity Assay Kit(Thermo Fisher Scientific). Sequencing is done on a MiSeq desktopsequencer using the MiSeq Reagent Kit V3 (lllumina) for 2× 300 bppaired-end sequencing. The 64-bit version of USEARCH (Edgar, 2013) isused for bioinformatical analysis of the sequence data.

In the HMO treated mice, VRE colonisation is reduced to undetectablelevels within 14 days. The density of VRE reduces within 5 days. Theuntreated mice continue to harbour large numbers of VRE in the colon.The treatment groups of mice also show an abundance ofPorphyromonadaceae, especially Barnesiella.

The invention claimed is:
 1. A process for obtaining the mixture ofhuman milk oligosaccharides (HMOs) consisting essentially of componentsA, B, C, and D: wherein the component A is 3-fucosyllactose ordifucosyllactose; the component B is selected from the group consistingof lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, and2′-fucosyllactose; the component C is lacto-N-fucopentaose II when thecomponent B is lacto-N-tetraose, lacto-N-fucopentaose III when thecomponent B is lacto-N-neotetraose, lacto-N-difucohexaose I when thecomponent B is lacto-N-fucopentaose I, or difucosyllactose when thecomponent B is 2′-fucosyllactose; and the component D is lactose whenthe component A is 3-fucosyllactose, or 2′-fucosyllactose when thecomponent A is difucosyllactose, wherein the component A is3-fucosyllactose when the component B is 2′-fucosyllactose; wherein theprocess comprises reacting the component A and the component B in thepresence of an α1-3/4 transfucosidase to produce a reaction mixture, andthen removing the α1-3/4 transfucosidase from the reaction mixture; andwherein either the component A is difucosyllactose and the processcomprises reacting difucosyllactose and the component B in a molar ratioof 0.2-5 in the presence of an α1-3/4 transfucosidase, wherein thereaction has a conversion rate of at least 10%; or the component A is3-fucosyllactose, and the process comprises reacting 3-fucosyllactoseand the component B in a molar ratio of 0.2-5 in the presence of anα1-3/4 transfucosidase, wherein the reaction has a conversion rate of atleast 35%.
 2. The process according to claim 1, wherein the process isused to obtain the mixture wherein the component B is lacto-N-tetraoseor lacto-N-neotetraose, and the molar ratio of (a) 3-fucosyllactose tothe component C or (b) the component B to the component C is not morethan
 2. 3. The process according to claim 1, wherein the processcomprises reacting 3-fucosyllactose and the component B in a molar ratioof 0.33-3 in the presence of an α1-3/4 transfucosidase.
 4. A process forobtaining a mixture of human milk oligosaccharides (HMOs) consistingessentially of: 3-fucosyllactose; a component B selected from the groupconsisting of lacto-N-tetraose, lacto-N-neotetraose,lacto-N-fucopentaose I and 2′-fucosyllactose; and a component C which islacto-N-fucopentaose II when the component B is lacto-N-tetraose,lacto-N-fucopentaose III when the component B is lacto-N-neotetraose,lacto-N-difucohexaose I when the component B lacto-N-fucopentaose I, ordifucosyllactose when the component B is 2′-fucosyllactose; andcomprising reacting 3-fucosyllactose and the component B in the presenceof an α1-3/4 transfucosidase to produce a reaction mixture, and thenremoving lactose and the α1-3/4 transfucosidase from the reactionmixture; wherein the process comprises reacting 3-fucosyllactose and thecomponent B in a molar ratio of 0.2-5 in the presence of an α1-3/4transfucosidase, wherein the reaction has a conversion rate of at least35%.
 5. The process according to claim 4, wherein the mixture of HMOsconsists essentially of: 3-fucosyllactose; a component B selected fromthe group consisting of lacto-N-tetraose, lacto-N-neotetraose, andlacto-N-fucopentaose I; and a component C which is lacto-N-fucopentaoseII when the component E is lacto-N-tetraose, lacto-N-fucopentaose IIIwhen the component E is lacto-N-neotetraose, or lacto-N-difucohexaose Iwhen the component E is lacto-N-fucopentaose I.